Gaseous nitric oxide-sequestering products and processes of preparing same

ABSTRACT

A process of preparing articles having gaseous NO sequestered therein is disclosed, as well as articles prepared thereby. Also disclosed are articles having gaseous NO sequestered therewithin and having reduced amount of oxygen-containing and/or nitrogen-containing reactive species. Also disclosed are processes of preparing packaged articles having a non-gas permeable package that comprises gaseous NO therein and packaged articles made therefrom. Also disclosed are charging devices which can be utilized in the above-described processes. The articles prepared by the above-described processes are preferably medical devices such as indwelling catheters, intubation devices and tampons. Tampons having sequestered therein gaseous NO, uses thereof and processes of preparing same are also disclosed.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to medicalproducts and, more particularly, but not exclusively, to products havinggaseous nitric oxide (NO) sequestered therein, to processes and systemsfor producing such products and to uses thereof.

Infection is a constant risk to any healthy person, and poses even ahigher risk to hospitalized patients. Infections which are a result oftreatment in a hospital or a healthcare service unit are often termednosocomial infections. The risk of infection is further increased whenthe natural infection barriers of skin or other epithelial surfaces arebreached during a surgical procedure, and/or otherwise in cases wherebacteria normally present on the skin or in the air is allowed to accessthe interior surfaces of the body.

Nosocomial infections occur even when good hygiene and sterile techniqueis followed, although the incidence is reduced. Despite even the mostrigorous aseptic procedures, people cannot be completely isolated frominfectious agents, and the use of prophylactic antibiotics, whilereducing some pathogens, may allow other pathogens (e.g., resistantpathogens) to emerge.

Medical devices used in the care or treatment of people are sterilizedbefore use. While medical devices that comprise metal, glass or otherheat-stable materials can be treated with steam (autoclave) or hot air,the use of various polymers and plastics in medical devices may renderheat-dependent sterilization unsuitable. Heat-sensitive medical devicesare typically sterilized using gamma irradiation or sterilant gastechnologies employing ethylene oxide, chlorine dioxide, hydrogenperoxide, and the like, which generally do not require heat application.

However, regardless of the method of sterilization, the medical deviceis only sterile until it is exposed to a non-sterile environment. Oncethe medical device is exposed to a non-sterile environment, which, forexample, may occur after a package containing the medical device isopened in a non-sterile environment, microbial contaminants in the airor in the subject's body (e.g., skin or intestinal flora) can contactthe device. Under the generally warm and moist conditions found in asubject's body, the microbial contaminants can establish an infection.Contamination may even occur in a surgical environment.

Catheter-associated bacteriuria (CAB) is the most common nosocomialinfection worldwide. It accounts for up to 40% of hospital-acquiredinfections in the US each year. CAB carries with it a 2.8-fold increasedrisk of death and results in bacteremia in approximately 3% of patients,constituting a serious complication. Bacteriuria is typically caused bya single organism, with Escherichia coli (E. Coli) being the mostfrequently isolated species. Together, Escherichia coli and Pseudomonasaeruginosa account for over 39% of cases. As a result of the widespreaduse of urinary catheterization, CAB results in considerableantimicrobial use. According to some studies, the daily rate ofbacteriuria in catheterized patients ranges from 3 to 8%; and theincidence of bacteriuria is directly related to the duration ofcatheterization.

Catheter-associated urinary tract infection (CAUTI) is the mostprevalent form of CAB. The risk of acquiring CAUTI depends on the methodand duration of catheterization, the quality of catheter care, and hostsusceptibility.

CAB is difficult to treat with current antimicrobial strategies becauseof the antibiotic resistance biofilm that forms from free-floatingbacteria that adhere to the surface of catheters and colonize them.These biofilm bacteria are highly differentiated and extremely resistantto antibiotics. Biofilms are especially relevant in catheterization, asindwelling urinary catheters provide a surface for the attachment ofmicrobial host cell binding receptors that are recognized by bacterialadhesins. This in turn facilitates microbial adhesion and subsequentcolonization with uropathogens. Catheterization also disrupts theuroepithelial mucosa, exposing new binding sites for bacterial adhesins.Once biofilms are established they shed cells that seed other sectionsof the catheter and bladder. They also protect pathogenic bacteria fromantimicrobials and the host immune response.

Various approaches have been developed to prevent biofilm formation,including the application of antiseptic lubricating gels at the catheterinsertion point, the use of antireflux valves, and the application of ataped seal to the catheter drainage tubing junction [Stickler.Neurourology and Urodynamics 27: 748, 2008]. In addition to thesemeasures, catheter coatings are also being investigated to determinewhether they can inhibit the formation of biofilms. Some studies withanti-adherence agents (e.g., heparin) have shown promise [Stickler.Symposium series (Society for Applied Microbiology) 163S-70S, 2002;Tenke et al. Int J Antimicrob Agents 23 Suppl 1: 567-S74, 2004]. Otherstudies have antibiotic coating to eradicate bacteria. In addition,gendine-coated catheters, silver alloy-coated and nitric oxide-coatedcatheters have demonstrated inhibition of biofilm formation in somerecent studies [Regev-Shoshani et al. Antimicrob agents and chemother54: 273-279, 2010]; Hachem et al. Antimicrob agents and chemother 53:5145-5149, 2009].

Silver is a very effective antibacterial substance and silver alloycoated-catheters have been utilized in recent years in an effort toreduce infection rates. However, silver oxide catheters were notassociated with a statistically significant reduction in CAB, and othermeta-analyses have similarly concluded that silver oxide coatedcatheters are ineffective. Recent Infectious Diseases Society of America(IDSA) guidelines (2009) described the treatment effect observed withsilver alloy-coated catheters as being significantly smaller in morerecent studies than in earlier research. Bjarnsholt et al. [APMIS 115:921-928, 2007] demonstrated that up to one hundred times more silverwould be needed to achieve efficacy against biofilm organisms ascompared to planktonic organisms.

Antibiotic-coated catheters may reduce the risk of CAB by preventing ordelaying onset in hospitalized patients [Darouiche et al., J infectdiseases 176: 1109-1112, 1997; Stickler, 2008, supra], and havedemonstrated antimicrobial effects against bacteriuria pathogens inseveral in vitro studies [Johnson et al. Antimicrob agents and chemother43: 2990-2995, 1999; Darouiche et al., 1997, supra], and some in vivostudies also report positive effects [Jacobsen et al. Clinic MicrobiolReviews 21: 26-59, 2008].

Ciprofloxacin, gentamicin, norflaxin, nitrofurazone, and combinations ofcompounds such as chlorhexidine and protamine sulfate have beensuccessfully incorporated into catheter coatings [Jacobson et al., 2008,supra]. Nitrofurans have proven to be effective against a wide spectrumof gram-positive and gram negative bacteria, including a variety ofstrains of the common urinary pathogens. In vitro studies indicate thatnitrofurazone catheters might have a stronger antibacterial effect thansilver hydrogel catheters [Bjarnsholt et al. 2007, supra]. However,nitrofurans have been demonstrated to be ineffective against moststrains of Pseudomonas aeruginosa, and they do not inhibit viruses orfungi. Nitrofurazone catheters are also very expensive.

Bacterial vaginitis (BV) is the most common vaginal infection in womenof childbearing age, with a prevalence of approximately 30% (Allsworth &Peipert, 2007). BV has gained increasing attention as manyepidemiological studies have established its association with a widearray of infectious morbidities, such as increased susceptibility tosexually transmitted infections (including HIV and genital herpes), aswell as unfavorable pregnancy outcomes, in particular preterm birth[Nyirjesy, P. 2008. Infect Dis Clin N Am 22: pp. 637-652].

Vulvovaginal candidiasis (VVC) is the second most common cause ofvaginitis (after bacterial vaginitis), diagnosed in up to 40% of womenwith vaginal complaints [Achkar and Fries, 2010, Clinical MicrobiologyReviews, April 2010, Vol. 23, No. 2: pp. 253-273; Andersen et al., 2004,JAMA 291: pp. 1368-1379; Bauters et al., 2002, Am. J. Obstet. Gynecol.187: pp. 569-574). Approximately 75% of women experience at least oneoccurrence of VVC during their childbearing years, and half of thesewomen have at least one recurrence [Sobel, The Lancet. Volume 369, Issue9577, 9 June 2007-15 Jun. 2007: pp. 1961-1971].

VVC affects primarily healthy women, with risk factors for infectionincluding frequent sexual intercourse, receptive oral sex, high-estrogenoral contraceptives, condoms, and spermicides, hormone replacementtherapy, uncontrolled diabetes mellitus, conditions with highreproductive hormone levels, genetic predispositions pregnancy, andantibiotic usage [Achkar and Fries, 2010, supra].

C. albicans is the most common species identified in VVC cases, with anestimated incidence of 76 to 89%. Candida albicans is an opportunisticfungus capable of colonizing the vaginal mucosa, which is present in thevaginal tracts of up to 70% of non-pregnant women [Bauters et al., 2002,supra; Beigi et al., 2004, Obstet. Gynecol. November; 104 (5 Pt 1):pp.926-30]. In the absence of immunosuppresion or compromised mucosa,individuals with vaginal colonization of Candida are asymptomatic. Upondisturbance in the balance between colonization and the host, Candidacan cause vulvovaginal candidiasis (VVC), resulting in clinical signsand symptoms of inflammation [Achkar and Fries, 2010, supra]. Thetransition from asymptomatic colonization to symptomatic candidiasis mayalso result from factors that enhance fungus virulence (Cassone et al.,2007).

An estimated 80 to 90% of uncomplicated VVC cases may be successfullytreated with short-course or single-dose therapy with azoles, withflucanozole being the most widely used oral azole; treatment durationranges from 1-7 days, depending on the product [Sobel, 2007, supra].Studies of vaginal yeast isolates suggest that resistance of C. albicansto fluconazole ranges from 3.7% to 68.2% [Bauters et al., 2002, supra].While topical azoles are considered to be safe and generally welltolerated, 5 to 10% of patients experience a burning sensation with use[Sobel, 2007, supra; Nyirjesy, 2008, supra]. Studies also suggest thatmost patients prefer oral azole administration for its convenience andlack of local side effects and messiness [Tooley, 1985, Practitioner229: pp. 655-660]. However, orally-administered azoles such asfluconazole may be associated with side effects, such as GI intoleranceand headache, and sometimes rash and liver toxicity [Nyirjesy, 2008,supra].

While VVC is commonly managed with intravaginal azole regimens ofvarying duration, there is a growing trend toward shorter therapies,including single-dose treatments. However, women with chronic orpersistent yeast infections (RVVC) are less likely to respond to shortertreatment regimens, and symptomatic relapse occurs in more than half ofRVCC cases within a short time of treatment cessation.

There is also concern that repeated azole treatments may induce drugresistance, shifting the spectrum of causative Candida species towardthe azole resistant non-C. albicans.

Multiple-day treatment regimens are more effective in women prone toRVVC and in certain populations, but the inconvenience of these regimensmay negatively impact treatment compliance and satisfaction, symptomcontrol, and quality of life. Furthermore, single-dose oral orintravaginal therapy is limited in its efficacy to the treatment of mildto moderate infections. Oral azoles are also contraindicated duringpregnancy, are ineffective in vaginitis caused by C. glabrata, and maybe associated with side effects such as GI intolerance, headache, andmore rarely, rash and liver toxicity.

Bacterial vaginosis (BV) is recognized as being the most common cause ofvaginal discharge. BV typically results from anaerobic bacterialinfections from species such as Gardnerella vaginalis, Mycoplasmahominis, and Ureaplasma urealyticum, although aerobic bacteria have alsobeen observed as causative agents.

To date, treatment of BV includes both intravaginal and oralformulations of the antibiotics clindamycin and metronidazole [Chen etal. J Womens Health (Larchmt). 18(12):1997-2004], although a cure rateof only 61% following metronidazole treatment has recently been reported[Mikic and Budakov, 2010. Arch Gynecol Obstet 282:43-47]. Furthermore,reported recurrence rates following antibiotic therapy range from 3 to58%. High rates of recurrence and lack of symptom resolution indicatethat the primary treatment of BV with antibiotics may not be completelyeffective. In addition, antibiotic resistance becomes a potential issuefacing continued antibiotic treatment of BV, as well as possible changesto the composition of the normal flora of the vagina.

Forty-two percent of women with recurrent vaginal infections have beenreported to resort to alternative therapies, such as probiotics orhomeopathy, dietary restriction of sugars and inclusion of yogurt, andhormonal manipulation with depot medroxyprogesterone and desensitizationtherapy, to prevent recurrences, yet, support for the efficacy of thesemethodologies are sparse and often lack methodological quality and/orsufficient data.

The feasibility of utilizing tampons as drug delivery systems forprolonged intravaginal drug administration of metronidazole has beenstudied using different commercially available tampon brands [Chien etal. J. Pharm. Sci. 71(7):767-771].

Nitric oxide (NO) is a small, naturally produced, hydrophobic,free-radical gas that has a major role in innate immunity. NO exhibitsbroad reactivity and rapid diffusive properties through biologicalliquids and lipid membranes, with a short half-life in a physiologicalmilieu [Subczynski and Wisniewska. 2000. Acta Biochim. Pol. 47:613-625].Overproduction of NO induced by the enzymatic activity of induciblenitric oxide synthase (iNOS) in various cell types has been shown toplay a vital role in several inflammatory and immunoregulatoryprocesses. NO has been shown to play important roles in vasodilatation,neurotransmission, angiogenesis, modulation of wound healing, andnonspecific responses to infection).

NO has been shown to be bacteriostatic and bactericidal. Miller et al.[Nitric Oxide 20: 16-23, 2009] demonstrated that multiple 30 minutetreatments of 160 ppm nitric oxide resulted in over a 5 log 10 colonyforming unit per milliliter (CFU/ml) decrease in the bacterial load ofStaphylococcus aureus, E. coli and Pseudomonas aeruginosa.

Nitric oxide is also known to function as an antimicrobial agent.Gaseous nitric oxide at a concentration of about 200 ppm has beendemonstrated to clear pneumonia caused by pathogens such as Pseudomonasaeruginosa or S. aureus [McMullin et al., 2005, Respir. Care5:1451-1456]. Topical applications of gaseous nitric oxide at about 200ppm has been demonstrated to inhibit or prevent growth of a variety ofmicrobial pathogens including P. aeruginosa, S. aureus, E. coli,Streptococcus spp. and Candida albicans [Ghaffari et al., 2005, NitricOxide, 14:21-29]. Nitric oxide has also been demonstrated to inhibitreplication of a variety of viral pathogens including influenza virus,retroviruses, rhabdoviruses e.g. vesicular stomatitis virus, flaviviruse.g. Japanese Encephalitis virus [Rimmelzwaan et al., 1999, J. Virol73(10): 8880-8883 and references cited therein].

At very low concentrations (up to 0.1 parts per million in air), gaseousnitric oxide (gNO) may be administered to humans having breathingproblems since it was found to have beneficial effects due to itsbronchodilatory and vasodilatory activity. However, nitric oxide israther complicated for use as a gas. Moreover, colorless gaseous NO mayreact with oxygen under certain conditions, yielding nitrogen dioxide(NO₂), a reddish-brownish gas with much higher toxicity than NO.

A variety of methodologies have been attempted to deliver and study NOtherapeutically. These include the use of polymers containing adiazeniumdiolate NO donor [Hetrick and Schoenfisch. 2007. Biomaterials28:1948-1956; Nablo et al. 2005. Biomaterials 26:6984-6990], exposurechambers for direct topical application of NO [Ghaffari et al. 2006.Nitric Oxide 14:21-29; McMullin et al. 2005. Respir. Care 50:1451-1456],and filling a urinary catheter retention balloon with nitrite andascorbic acid to release NO [Carlsson et al. 2005. Antimicrob. AgentsChemother. 49:2352-2355].

Nitric oxide-producing agents have been used to keep various medical andother devices substantially free of bacteria by their capacity torelease nitric oxide under certain conditions. For example, U.S. Pat.Nos. 5,525,357, 7,105,502, 7,122,529 and 7,829,553, U.S. PatentApplication Nos. 2003/1039697, 2004/0247640, 2006/0008529 and20070196327, and International Patent Application Nos. WO 1995/024908,WO 2006/037105, WO 2007/1028657, WO 2002/1056864, WO 2006/1084910 and WO2006/1078786, teach various industrial materials and polymers comprisingnitric oxide precursors, which are capable of generating some amount ofNO in situ when the material comes in contact with physiological media,or upon other chemical or physical stimuli.

Additional background art includes U.S. Pat. Nos. 5,374,710, 5,155,137,6,951,902, 6,949,530, 6,911,478 and 6,110,453.

U.S. Patent Application Nos. 2010/0268149 and 2011/0076313, and WO2009/036571 teach nitric oxide gas-releasing conduit configured forsurgical implantation through a patient's tympanic membrane.

Regev-Shoshani et al. (2010, supra) disclose studies conducted forinvestigating the potential of NO to prevent biofilm formation.Catheters impregnated with gaseous nitric oxide (gNO) demonstratedslow-release of nitric oxide over a 14-day period, were renderedantiseptic, and were able to prevent bacterial colonization and biofilmformation on their luminal and exterior surfaces. NO impregnatedcatheters inhibited the growth of Escherichia coli within thesurrounding media and eradicated bacterial concentrations of up to 10⁴CFU/ml.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing an article having gaseousnitric oxide sequestered therewithin, the process comprising: placing anarticle within a chamber; generating a reduced pressure in the chamber;and filling the chamber with a gaseous nitric oxide-containingenvironment, thereby preparing the article having gaseous nitric oxidesequestered therewithin.

According to some embodiments of the invention, generating the reducedpressure comprises reducing the pressure by from −1 psi to −50 psi.

According to some embodiments of the invention, reducing the pressure issuch that a humidity in the article is reduced by at least 50%.

According to some embodiments of the invention, reducing the pressure issuch that an amount of oxygen in the article is reduced by at least 50%

According to some embodiments of the invention, filling the chamber withthe gaseous nitric oxide-containing environment is effected for a timeperiod that ranges from 30 minutes to 24 hours.

According to some embodiments of the invention, the article includes atleast a portion of a surface configured to sequester nitric oxide.

According to some embodiments of the invention, the article includes atleast a portion of a surface configured to sequester at least 1 ppmnitric oxide per cm³.

According to some embodiments of the invention, the article includes atleast a portion of a surface configured to sequester at least 200 ppmnitric oxide per cm³.

According to some embodiments of the invention, at least a portion ofthe article comprises a plurality of voids for sequestering nitricoxide.

According to some embodiments of the invention, the article is a medicaldevice.

According to some embodiments of the invention, the medical device isselected from the group consisting of a catheter, an endotracheal tube,a tampon, a tubing, a prosthetic, a medical implant, an artificialjoint, an artificial valve, a needle, an intravenous access device, acannula, a biliary stent, a nephrostomy tube, a vascular graft, aninfusion pump, an adhesive patch, a suture, a fabric, a mesh, apolymeric surgical tool or instrument, an intubation device,prosthetics, artificial joints, artificial valves, adhesive patches,sutures, fabrics, a cardiovascular stent, a cardiac surgery device, anorthopedic surgery device, an orthodontic or periodontic device, adental surgery device, a veterinary surgery device, a bone scaffold, ahemodialysis tubing or equipment, a blood exchanging device, animplantable prostheses, a bandage, a heart valve, an ophthalmic deviceand a breast implant.

According to some embodiments of the invention, the medical device is animplantable device.

According to some embodiments of the invention, the medical device isselected from the group consisting of an indwelling catheter and atracheal tube.

According to some embodiments of the invention, the catheter is selectedfrom the group consisting of urinary catheter, central venous catheter,biliary vascular catheter, pulmonary artery catheter, peripheral venouscatheter, arterial line, central venous catheter, peritoneal catheter,epidural catheter and central nervous system catheter.

According to some embodiments of the invention, the medical device is atampon.

According to some embodiments of the invention, the medical devicecomprises a polymeric material.

According to some embodiments of the invention, the polymeric materialis a hydrophobic polymeric material.

According to some embodiments of the invention, the polymeric materialis selected from the group consisting of silicone, polyacetal,polyurethane, polyester, polytetrafluoroethylene, polyethylene,polymethylmethacrylate, polyhydroxyethyl methacrylate, polyvinylalcohol, polypropylene, polymethylpentene, polyetherketone,polyphenylene oxide, polyvinyl chloride, polycarbonate, polysulfone,acrylonitrile-butadiene-styrene, polyetherimide, polyvinylidenefluoride, polysiloxane, fluorinated polysiloxane, polyanhydride,ethylene vinyl acetate, methacrylic acid, ethylene oxide, propyleneoxide, polystyrene, ethylene-propylene rubber, fluoroelastomer, silasticelastomer, polyethylene tetrephthalate, colloidion, carbothane, nylon,poly(L-lactide), poly(DL-lactide), poly(DL-lactide-co-glycolide),poly(e-caprolactone), polyparadioxanone, polytrimethylene carbonate,collagen, silk, elastin, chitin, coral, hyaluronic acid, bone, rayon,cotton, cellulosic polymer, copolymers of any of forgoing and anycombination of the forgoing.

According to some embodiments of the invention, the polymeric materialis a hydrophilic or amphiphilic polymeric material.

According to some embodiments of the invention, the polymeric materialis selected from the group consisting of rayon, cotton and cellulosicpolymer (as in the case of, for example, tampons).

According to some embodiments of the invention, the article is selectedfrom the group consisting of a packaged article and a bare (unpackaged)article.

According to some embodiments of the invention, the packaged articlecomprises a gas-permeable package.

According to some embodiments of the invention, placing the article inthe chamber comprises positioning the article within an enclosure.

According to some embodiments of the invention, the enclosure is anon-gas permeable enclosure.

According to some embodiments of the invention, the process furthercomprises, subsequent to the filling, sealing the enclosure.

According to some embodiments of the invention, the process furthercomprises disposing a desiccant within the enclosure.

According to some embodiments of the invention, the process furthercomprises disposing a nitric oxide indicator within the enclosure.

According to as aspect of some embodiments of the present inventionthere is provided an article having a gaseous nitric oxide sequesteredtherewithin, prepared by the process described hereinabove.

According to some embodiments of the invention, the article furthercomprises an enclosure.

According to as aspect of some embodiments of the invention there isprovided an article having sequestered therewithin at least 1 ppm nitricoxide per cm³ and comprising less than 1 ppm per cm³ nitrogen-containingand/or oxygen containing reactive species.

According to some embodiments of the invention, the article hassequestered therein from 1 ppm to 200 ppm per cm³ nitric oxide.

According to some embodiments of the invention, the sequestered nitricoxide is releasable in an aqueous solution during a time period thatranges from 1 hour to 1 month.

According to some embodiments of the invention, any of the articlesdescribed herein further comprises an enclosure.

According to some embodiments of the invention, the enclosure comprisesa gaseous nitric oxide-containing environment.

According to some embodiments of the invention, the environment is anambient environment.

According to some embodiments of the invention, the enclosure is anon-gas permeable enclosure.

According to some embodiments of the invention, any of the articlesdescribed herein further comprises a desiccant disposed within theenclosure.

According to some embodiments of the invention, any of the articlesdescribed herein further comprises a nitric oxide indicator disposedwithin the enclosure.

According to some embodiments of the invention, any of the articlesdescribed herein comprises at least 1 ppm per cm³ nitric oxidesequestered therewithin.

According to some embodiments of the invention, any of the articlesdescribed herein comprises at least 200 ppm per cm³ nitric oxidesequestered there within.

According to some embodiments of the invention, the sequestered nitricoxide is releasable is an aqueous solution during a time period thatranges from 1 hour to 1 month.

According to some embodiments of the invention, any of the articlesdescribed herein is being substantially devoid of humidity.

According to some embodiments of the invention, any of the articlesdescribed herein is being substantially devoid of oxygen.

According to some embodiments of the invention, an amount ofnitrogen-containing and/or oxygen containing reactive species in thearticle is lower than 1 ppm per cm³.

According to some embodiments of the invention, an amount ofnitrogen-containing and/or oxygen containing reactive species in thearticle is lower than 1 ppb per cm³.

According to some embodiments of the invention, any of the articlesdescribed herein is a medical device, as described herein.

According to an aspect of some embodiments of the present inventionthere is provided a tampon having sequestered therein gaseous nitricoxide.

According to some embodiments of the invention, an amount of the nitricoxide ranges from 1 ppm to 200 ppm per cm³.

According to some embodiments of the invention, an amount of the nitricoxide is at least 200 ppm per cm³.

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a tampon having sequesteredtherein nitric oxide, the process comprising: exposing a tampon togaseous nitric oxide-containing environment, thereby preparing thetampon having sequestered therein nitric oxide.

According to some embodiments of the invention, the exposing comprises:placing the tampon is a chamber; and filling the chamber with the nitricoxide-containing environment.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, generating a reduced pressure in thechamber.

According to an aspect of some embodiments of the invention there isprovided a method of treating a vaginal medical condition in a subjectin need thereof, the method comprising placing a tampon having gaseousnitric oxide sequestered therein in a vagina of the subject.

According to an aspect of some embodiments of the invention there isprovided a tampon having gaseous nitric oxide sequestered therein,identified for use in the treatment of a vaginal medical condition.

According to an aspect of some embodiments of the invention there isprovided a method of treating a vaginal medical condition, the methodcomprising intravaginally administering to a subject in need thereofgaseous nitric oxide.

According to some embodiments of the invention, intravaginallyadministering gaseous nitric oxide comprises placing a tampon havinggaseous nitric oxide sequestered therein.

According to an aspect of some embodiments of the invention there isprovided gaseous nitric oxide, for use in the treatment of a vaginalmedical condition.

According to an aspect of some embodiments of the invention there isprovided a use of gaseous nitric oxide in the manufacture of amedicament for treating a vaginal medical condition.

According to some embodiments of the invention, the gaseous nitric oxideis being sequestered in a tampon.

According to an aspect of some embodiments of the invention there isprovided a tampon having gaseous nitric oxide sequestered therein, foruse in delivering gaseous nitric oxide to a vagina of a subject.

According to an aspect of some embodiments of the invention there isprovided a process of preparing a packaged article, wherein the packagedarticle comprises a gas-permeable package, the process comprising:exposing a packaged article to a gaseous nitric oxide-containingenvironment, thereby preparing the packaged article.

According to some embodiments of the invention, the exposing comprises:placing the packaged article in a chamber; and filling the chamber withthe nitric oxide-containing environment.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, sealing the chamber.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, generating a reduced pressure in thechamber.

According to some embodiments of the invention, generating the reducedpressure is such that a humidity in the enclosure is reduced by at least50%.

According to some embodiments of the invention, reducing the pressure issuch that an amount of oxygen in the enclosure is reduced by at least50%.

According to some embodiments of the invention, filling is effected byflowing nitric oxide into the chamber.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, absorbing humidity from the package.

According to some embodiments of the invention, filling is effected suchthat an ambient environment is provided within the package.

According to some embodiments of the invention, the nitricoxide-containing environment comprises at least 0.01% nitric oxide.

According to some embodiments of the invention, the packaged article hasgaseous nitric oxide sequestered within the article.

According to an aspect of some embodiments of there invention there isprovided a process of preparing a packaged article, wherein the packagedarticle comprises a non-gas permeable enclosure, the process comprising:positioning an intact article within the non-gas permeable enclosure, tothereby obtain a non gas-permeable enclosure having the article disposedtherewithin; exposing the enclosure with a gaseous nitricoxide-containing environment, so as to introduce into the enclosure thenitric oxide-containing environment; and sealing the enclosure, therebypreparing the packaged article.

According to some embodiments of the invention, the exposing comprises:placing the enclosure in a chamber; and filling the chamber with thegaseous nitric oxide-containing environment.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, sealing the chamber.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, reducing a pressure in the chamber.

According to some embodiments of the invention, reducing the pressure issuch that a humidity in the enclosure is reduced by at least 50%.

According to some embodiments of the invention, reducing the pressure issuch that an amount of oxygen in the enclosure is reduced by at least50%.

According to some embodiments of the invention, the filling is effectedby flowing nitric oxide into the chamber.

According to some embodiments of the invention, the process furthercomprises, prior to the filling, absorbing humidity from the enclosure.

According to some embodiments of the invention, the filling is effectedsuch that an ambient environment is provided within the enclosure.

According to some embodiments of the invention, the nitricoxide-containing environment comprises at least 0.02% nitric oxide.

According to some embodiments of the invention, the packaged article hasgaseous nitric oxide sequestered within the article.

According to an aspect of some embodiments of the invention there isprovided a packaged article prepared by the process described herein.

According to an aspect of some embodiments of the invention there isprovided a package comprising: a material configured to form anenclosure; a article disposed within the enclosure; and a gaseous nitricoxide-containing environment within the enclosure.

According to some embodiments of the invention, the package is a non-gaspermeable package.

According to some embodiments of the invention, the enclosure is asealed enclosure.

According to some embodiments of the invention, the environment is anambient environment.

According to some embodiments of the invention, the environmentcomprises gaseous nitric oxide in an amount sufficient to sterilize aninterior of the enclosure and the article.

According to some embodiments of the invention, the amount of gaseousnitric oxide is at least 200 ppm.

According to some embodiments of the invention, the article has gaseousnitric oxide sequestered therewithin.

According to some embodiments of the invention, the amount of thegaseous nitric oxide sequestered in the article is at least 1 ppm.

According to some embodiments of the invention, the gaseous nitric oxidesequestered in the article is releasable in an aqueous solution duringat least 1 minute.

According to some embodiments of the invention, the package furthercomprises a desiccant.

According to some embodiments of the invention, the package furthercomprises a nitric oxide indicator.

According to some embodiments of the invention, the article is a medicaldevice, as described herein.

According to an aspect of some embodiments of the present inventionthere is provided a charging device comprising: a chamber comprising aninlet for receiving a gaseous nitric-oxide containing environment and anoutlet for releasing the gaseous nitric-oxide containing environment;and an article disposed within the chamber.

According to some embodiments of the invention, the device furthercomprises a desiccant configured to absorb humidity from the gaseousenvironment.

According to some embodiments of the invention, the device furthercomprises a nitric oxide indicator configured to undergo a color changesuitable for visual assessment of whether the article has been exposedto the nitric oxide.

According to some embodiments of the invention, the device furthercomprises an outlet for generating a reduced pressure in the chamber.

According to some embodiments of the invention, the device furthercomprises a package enclosing the article.

According to some embodiments of the invention, package is a non-gaspermeable package.

According to some embodiments of the invention, the article includes atleast a portion of a surface configured to sequester the nitric oxide.

According to some embodiments of the invention, the surface isconfigured to sequester at least 1 ppm nitric oxide.

According to some embodiments of the invention, the surface isconfigured to sequester about 1 ppm nitric oxide to about 20,000 ppmnitric oxide.

According to as aspect of some embodiments of the present inventionthere is provided a charging device comprising: a sealed chamber havinga reduced pressure therewithin; and an article disposed within thechamber.

According to some embodiments of the invention, the chamber furthercomprises an outlet for generating the negative pressure within thechamber.

According to some embodiments of the invention, the device furthercomprises an inlet configured for receiving a gaseous nitricoxide-containing environment and an outlet for releasing the gaseousnitric oxide-containing environment.

According to some embodiments of the invention, the device furthercomprises a package enclosing the article.

According to some embodiments of the invention, the package is selectedfrom the group consisting of a gas permeable package and a nongas-permeable package.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a bar graph demonstrating the antimicrobial effect of anexemplary polymeric medical device sample (a catheter section) dosedwith nitric oxide for 1, 5, 15, 30, 60 or 120 minutes, upon incubationin a bacterial suspension of 10³, 10⁵ or 10⁸ CFU/ml for 8 hours at 37°C., while showing the number of CFU/ml remaining in the suspension withrespect to dose time and starting culture CFU/ml.

FIG. 2 is a chart presenting the total accumulation of nitrites andnitrates, produced from an exemplary medical device sample (a cathetersection) dosed with nitric oxide and immersed in water, and showing atime-dependent nitric oxide dosing.

FIGS. 3A-B present a micrograph (FIG. 3A) and bar graphs (FIG. 3B)demonstrating the effect of catheter storage conditions on theantimicrobial activity of control and nitric oxide-eluting samples afterstorage and upon incubation with suspensions of 10³ CFU/ml E. Coli for 1minute followed by 24 incubation in PBS. FIG. 3A presents images ofrepresentative three-compartment petri plates of control and nitricoxide-eluting samples after both sets were stored for 7 days in 50 mlsterile air (I) or 5 ml sterile water (II); FIG. 3B presents the viablecounts of the triplicate CFU with the hatched bars are data from thecontrol, and the checkered bars are data from the NO-impregnatedcatheters. The error bars indicate standard deviations.

FIGS. 4A-B present a bar graph (FIG. 4A) and a micrograph (FIG. 4B)showing the biofilm formation and biofilm-embedded bacteria on the innerlumen of control and NO-dosed catheter samples subjected to 24 hours ofurine flow. FIG. 4A presents biofilm formation as the absorbance at 595nm of the crystal violet attached to the catheter pieces afterextraction with ethanol. The hatched bars are data from the control,while the checkered bars are data from the impregnated catheters. FIG.4B presents representative images of biofilm-embedded bacteriadetermined in 1 μl (bottom) and 10 μl (top) of water surrounding aselected catheter piece and incubated for 24 hours.

FIGS. 5A-B present a photograph showing the effect of nitric oxide gason biofilms of Escherichia coli after exposure for 5, 10, 30, 60 or 120minutes, relative to controls (FIG. 5A); and a photograph showing theeffect of nitric oxide gas on biofilms of Acinetobacter baumanii afterexposure for 5, 10, 30, 60 or 120 minutes, relative to controls (FIG.5B).

FIG. 6 presents a bar graph showing the antimicrobial activity of nitricoxide-dosed catheter sections on the bacterial strains Enterococcusfaecalis #29212 (E.f. #29212), Staphylococcus saprophyticus #15305 (S.s#15305), Staphylococcus epidermidis #35984 (S.e. #35984), Escherichiacoli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210 (P.a. #14210),Acinetobacter baumanii #BAA-747 (A.b. #BAA-747 and, Candida albicans(C.a. #14053).

FIG. 7 is a bar graph showing the antimicrobial activity of nitricoxide-dosed catheters the bacterial clinical isolates Enterococcusfaecalis (E.f.), Staphylococcus aureus (S.a.), E. coli (E.c.), P.aeruginosa (P.a.), and Stenotrophomonas maltophilia (S.m.).

FIG. 8 is a bar graph presenting the relative biofilm formation onluminal surfaces of nitric oxide-sequestering catheter sectionsfollowing 72 hour incubation in urine inoculated with 10³ CFU/ml ofEnterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus#15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984),Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210(P.a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA-747), andCandida albicans (C.a. #14053).

FIG. 9 presents a bar graph showing the data obtained for the growth ofbiofilm-embedded bacteria on nitric oxide-sequestering catheter sectionsfollowing 72 hours incubation in urine inoculated with 10³ CFU/ml ofEnterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus#15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984),Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210(P.a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA-747), andCandida albicans (C.a. #14053).

FIGS. 10A-C present scanning electron micrographs of Staphylococcusepidermidis (ATCC #35984) biofilms on Untreated (control) cathetersections, magnification 2.5 k (inset=20.0 k) (FIG. 10A); Untreated(control), magnification 2.5 k (right inset=15 k, left inset=20 k) (FIG.10B); and NO-sequestering catheter sections, magnification 1.5 k (FIG.10C).

FIGS. 11A-C present scanning electron micrographs of A. baumanii (ATCC#BAA-747) biofilms on Untreated (control) catheter sections,magnification 2.5 k (inset=15.0 k) (FIG. 10A); Untreated (control),magnification 1.0 k (inset=5.0 k) (FIG. 10B); and NO-sequesteringcatheter sections, magnification 1.0 k (FIG. 10C).

FIG. 12 is a bar graph presenting comparative data of E. coli growth inmedia containing pieces of an NO-sequestering catheter (NOX), asilver-alloy coated catheter (AG) and an antibiotic-coated catheter(NFC) versus media from control catheter, after immersion of thecatheters for 24 hours in suspension comprising 10³ CFU/ml and incubatedfor 24 hours at 37° C.

FIG. 13 presents images of representative three compartment LB agarpetri plates showing E. coli growth in urine after 72 hours exposure topieces of an NO-sequestering catheter (NOX; bottom left), a silver-alloycoated catheter (AG; top right) and an antibiotic-coated catheter (NFC;bottom right) and control catheters (top left). Within each threecompartment LB agar petri plate 1, 10, and 100 μl of each sample wereplated and incubated overnight at 37° C.

FIG. 14 presents images of representative three compartment LB agarpetri plates showing E. coli colonization on NO-sequestering catheter(NOX; bottom left), a silver-alloy coated catheter (AG; top right) andan antibiotic-coated catheter (NFC; bottom right) and control catheters(top left), after immersion of catheters for 24 hours in suspensioncontaining 10³ CFU/ml of E. coli. In each LB Petri dish a catheter wasrolled over the surface and then incubated at 37° C. overnight.

FIGS. 15A-B present bar graphs showing comparative data of colonizedbiofilm formation on NO-sequestering catheter (NOX), a silver-alloycoated catheter (AG) and an antibiotic-coated catheter (NFC) versuscontrol after 72 hours of incubation, demonstrated by absorbance at 595nm (FIG. 15A) and the bacterial growth from the biofilms from thedifferent catheters (FIG. 15B).

FIG. 16 presents comparative plots showing nitrite release over timefrom the NO-sequestering Tracheal tubes Mallinckrodt: Hi-Lo Trachealtube 6.5 mm ID ref No. 86110 (triangles) and Mallinckrodt: Hi-ContourTracheal tube 4.5 oral/nasal 6.2, 11 mm ID ref No. 107-45.

FIG. 17 is a bar graph showing the amount of nitric oxide released after30 minutes from four exemplary NO-sequestering tampons (see, Table 19),as measured by the respective total amount of nitrites released from thetested tampons Nitrites were measured using Griess reagent. The nitritesreleased were calculated per 1 tampon.

FIG. 18 presents comparative plots showing the accumulation profiles ofnitric oxide production during the first 5 hours for four exemplaryNO-impregnated tampons, as measured by the respective total accumulationof nitrites in water produced from the tested tampons. Nitrites weremeasured using Griess reagent. The nitrites released were calculated per1 tampon

FIGS. 19A-D demonstrate the antimicrobial effect of four exemplaryNO-sequestering tampons A (FIG. 19A), B (FIG. 19B), C (FIG. 19C) and D(FIG. 19D), as identified in Table 19 herein, by showing images ofrepresentative petri plates in which NO-treated tampons inoculated withC. albicans culture for 4 hours at 30° C. were rolled and uponincubating the plates overnight at 30° C.

FIG. 20 is a bar graph demonstrating the anti-infective activity of fourexemplary NO-sequestering tampons A-D, as identified in Table 19 herein,by showing the growth of C. albicans in media after immersion of thetampons for 6 hours in suspension comprising 10¹ CFU/ml of C. albicans(dark bars) compared with negative control tampons (white bars). Numbersrepresent viable counts of the triplicate CFUs. Error bars representstandard deviation.

FIG. 21 is a bar graph demonstrating the anti-infective activity of fourexemplary NO-sequestering tampons A-D, as identified in Table 19 herein,by showing the growth of C. albicans in media after immersion of thetampons for 4 hours in suspension comprising 10² CFU/ml of C. albicans(dark bars) compared with negative control tampons (white bars). Numbersrepresent viable counts of the triplicate CFUs. Error bars representstandard deviation.

FIG. 22 presents representative photos demonstrating the zone ofinhibition resulting from NO-treated tampons. NO-treated tampon B (topright), NO-treated tampon C (bottom right) and untreated tampons B and C(top and bottom left, respectively) were cut lengthwise and placed ontoa LB agar petri plate that had been inoculated with 200 μl of E. coli at10⁶ CFU/ml, then incubated overnight at 37° C.

FIG. 23 presents a schematic illustration of the experimental setup usedto test the zone of microbial inhibition in a vaginal model.

FIG. 24 presents representative photos demonstrating the zone ofinhibition in a vaginal model. A solution containing LB and 2% (w/v)gelatin was inoculated with E. coli (10⁶ CFU/ml), placed in a 250 mlErlenmeyer flask and tampon B (see, Table 19) was suspended therein. TheErlenmeyer flasks were incubated at 37° C. overnight. Shown is a pictureof NO-treated (right) and untreated (left) tampon B placed inside theinoculated solution after incubation.

FIG. 25 shows total accumulation of nitrites, produced in water during 4hours, from tampons impregnated with nitric oxide outside theirwrappers. Nitrites were measured using Griess reagent. The nitritesreleased were calculated per 1 tampon

FIG. 26 presents a schematic illustration of an exemplary chargingdevice 300, according to some embodiments of the present invention,which is fitted with an inlet 345 for receiving gNO within chamber 337to form gaseous nitric oxide-containing environment 306, valve 315 forclosing outlet 345, an outlet 326 for releasing ambient environment orgaseous nitric oxide-containing environment from chamber 337, valve 336for closing outlet 326, an article 301 disposed within chamber 337, andan optional moisture scavenger in the form of desiccant 302 disposedwithin chamber 337 and configured to absorb an amount of humidity fromthe within chamber 337;

FIG. 27 is a schematic illustration of an exemplary charging device 300,as presented in FIG. 26, wherein chamber 337 also includes a surfacecoating 370 which protects the material of chamber 337 from chemicallyinteracting with gNO-containing environment 306, a gNO indicator 371configured to undergo a color change when article 301 has been exposedto gNO in environment 306, and humidity indicator 374 for signalingexposure of article 301 to humidity;

FIG. 28 is a schematic illustration of an exemplary charging device 400,according to some embodiments of the present invention, which is fittedwith an inlet 445 for receiving gNO within chamber 447 to form gaseousnitric oxide-containing environment 406, valve 415 for closing outlet445, an outlet 426 for releasing ambient environment or gaseous nitricoxide-containing environment from chamber 447, valve 436 for closingoutlet 426, and a non-gas permeable package 473 disposed within chamber447 and housing article 401;

FIG. 29 is a schematic illustration of an exemplary charging device 400,as presented in FIG. 28, wherein chamber 437 includes surface coating470 which protects the material of chamber 437 from chemicallyinteracting with gNO-containing environment 406, a gNO indicator 471configured to undergo a color change when article 401, havinggNO-sequestering surface 472 and housed within non-gas permeable package473 equipped with seal 490, has been exposed to gNO in environment 406,and humidity indicator 474 for signaling exposure of article 401 tohumidity;

FIG. 30 presents a schematic illustration of an exemplary chargingdevice 500 having a chamber 137, inlet 125 with valve 11, inlet 135 withvalve 2, and inlet 145 with valve 15, inlets which may share a commontube 23 with valve 19 that connects to chamber 137; outlet 126 withvalve 36 and connect to purge flow rotomoter 46, outlet 136 with valve41 and connect to purge flow rotomoter 45, outlets which may share acommon tube 33 that connects to chamber 137; and

FIGS. 31A-B present schematic illustrations of a package for chargingand containing gNO-sequestering medical device, according to someembodiments of the present invention, such as a package 200, comprisingnon-gas permeable package 203 configured to be impermeable to gNO andoptionally comprises seal 205 (FIG. 31B), medical device 201 disposedwithin non-gas permeable package 203, gNO-containing environment 206engulfing medical device 201 within non-gas permeable package 203 andcomprising a predetermined concentration of gNO capable of sterilizingthe interior of non-gas permeable package 203 and medical device 201,and further optionally comprising nitric oxide indicator 204 (FIG. 31B),desiccant 202 disposed within non-gas permeable package and configuredto absorb humidity from gNO-containing environment 206, and humidityindicator 208 (FIG. 31B) configured to indicate moisture saturation ofthe desiccant 202.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to medicalproducts and, more particularly, but not exclusively, to products havinggaseous nitric oxide (NO) sequestered therein, to processes and systemsfor producing such products and to uses thereof.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples. The invention iscapable of other embodiments or of being practiced or carried out invarious ways.

As discuss hereinabove, impregnation of gaseous nitric oxide (gNO) inmedical devices is regarded highly beneficial for sterilizing articlessuch as medical devices, by imparting the medical devices with the gNOcapabilities to reduce or prevent growth of a variety of pathogenicmicroorganisms, as well as biofilm formation.

As demonstrated in the Examples section that follows, gaseous nitricoxide sequestered in the solid substance of medical devices, can be usedto kill bacteria in an established biofilm and in the surroundingenvironment; be used as an effective sterilizing agent of such devices;and prevents adherence and growth of various strains or species ofbacteria and fungi over an extended period of time followingsterilization.

It has further been demonstrated that medical devices comprising one ormore polymers and sequestering gaseous nitric oxide are self-sterilizingat a sufficient level so as to prevent microbial adherence and growth onthe medical device or in the liquid media in its immediate vicinity foran extended period of time.

Furthermore, it has been demonstrated that the physical properties ofpolymeric material composing the device, as exemplified by silicone, orsilicone-coated medical devices, are not substantively altered bytreatment with nitric oxide, however, in some cases, a color change isobserved that may be useful as an indicator of NO load of the device.

Overall, impregnation of gNO in medical devices has been shown toeffectively provide the device with self-sterilization capacity.

As further demonstrated in the Examples section that follows, and isreferred to in more detail hereinunder, the present inventors havefurther practiced NO impregnation with medical devices such as tampons,and have shown that (i) tampons can be effectively charged with gNO soas to result in gNO-sequestering tampons; and (ii) tampons having gNOsequestered therewithin efficiently reduce microbial growth and thus canbe used to treat or prevent various common vaginal infections.

In view of the proven beneficial properties of medical devices havinggNO sequestered therewithin, the present inventors have searched for animproved process for impregnating gNO within medical devices. Thepresent inventors have thus designed and successfully practiced aprocess which involves exposing the article to be impregnated (e.g., amedical device) to reduced pressure, so as to substantially evacuateambient oxygen and humidity prior to exposure to gNO.

Without being bound by any particular theory, the present inventors havecontemplated that by removing ambient atmosphere from the article to beloaded, and thereby evacuating voids within the article, the gNOsequestering efficiency of the article would be improved due to enhancedfree void volume to be occupied by gNO. The enhancement in void volumecan be translated in some cases to enhanced affinity of NO, as isdiscussed in detail hereinunder. The present inventors have alsocontemplated that removal of ambient atmosphere would prevent gNOdegradation and would thus substantially reduce the production ofundesired reactive nitric oxide species associated with gNO degradation.

While reducing the present invention to practice it was found that theabove-described process substantially improved previous methodologies byimproving the efficiency by which gNO is sequestered from a nitricoxide-containing environment (gNO-containing environment), namely byimproving the gNO intake by an article which has been pre-exposed toreduced pressure prior to exposure to a gNO-containing environment. Byenhancing the relative amount of gNO taken from the gNO-containingenvironment, less gNO was spent during the loading process, and shorterexposure time was spent in order to achieve the same gNO loading levelscompared to some previously described processes. This process hastherefore been shown to be more efficient in terms of both time andcost, and to be safer, as compared to processes that involve loadingarticles with gaseous nitric oxide without pre-exposure to reducedpressure.

This process has further been shown efficient by substantially reducingthe formation of reactive species (e.g., reactive oxygen species) in thearticle, which bears a particular importance for implantable medicaldevices, as is discussed in further detail hereinunder.

The present inventors have further contemplated a process for producingsterilized and even self-sterilizing packages, which takes advantage ofthe sterilizing effect imparted by gaseous NO. Thus, the presentinventors have designed and successfully practiced a process in whichmedical devices are packaged in a non-gas permeable enclosure which isloaded with gaseous NO. Such a packaging process allows, for example,storing and/or transporting packaged articles while maintaining thepackaged articles sterilized.

Hence, according to an aspect of embodiments of the present invention,there is provided a process of preparing an article having gaseousnitric oxide (gNO) sequestered therewithin. The process, according tothis aspect of embodiments of the invention, is effected by:

placing an article within a chamber;

generating a reduced pressure in the chamber; and

filling the chamber with a gaseous NO-containing environment.

For clarity, it is noted that the term “sequestering” in the context ofgaseous nitric oxide (gNO), and the terms “impregnated”, “charged”,“loaded”, “dosed” and “treated” are used interchangeably hereinbelow andthroughout to denote an article releasably sequestering gNO therein.

As used herein, the term “sequestering” and any inflections thereofrefer to a state of an article having a foreign substance, such as agas, incorporated therein; a state which exists substantially from thetime the substance is introduced into the article from an externalsource to the time the substance leaves the article. According to someembodiments of the present invention, in the chemical sense thesequestered substance being released from the article is essentially thesame substance that was charged into the article.

The term “sequestering” therefore encompasses the phrase “releasablysequestering”.

Hence, “releasably sequestering”, as used herein, is meant to define anarticle having gNO absorbed therein in a reversible manner, wherein thegNO can be released to the ambient environment from the article undercertain conditions. According to some embodiments of the presentinvention, gNO is released from the article releasably sequestering gNOat an essentially controllable manner.

The phrase “sequestered therewithin” refers to gaseous NO sequesteredwithin the article, namely, in or on at least a portion of the articleor in or on any surface thereof. The terms “therewithin” and “therein:are used herein interchangeably.

In the context of embodiments of the invention, gNO is releasablysequestered in an article by charging the article with gNO.

As discussed in detail hereinbelow, the amount of sequestered gNOdepends, among other factors, on the size, volume, surface area andcomposition of the article. The article may be composed and assembledfrom various parts, layers and compositions, each having a differentcapacity to sequester gNO according to chemical composition,accessibility, surface area and the likes. In some embodiments, gNO isreleasably sequestered within at least a portion of the surface of thearticle.

For simplicity, a part of the article which can sequester gNO isreferred to herein as a “surface”, however it is to be understood thatthe term “surface” as used herein is not limited to the exterior orupper boundary of the article, not to an external part or a layer of thearticle, and not to its outwardly expressed manifestations of thearticle, but rather to all of these features as well as all otherinternal and external features of the article which can sequester gNO,according to some embodiments of the present invention.

In cases of articles having components which are inaccessible to thegNO-containing environment, the article can be disassembled to exposethese components, or be assembled from raw materials and/or componentsthat were charged with gNO according to some embodiments of the presentinvention.

It is noted herein that the article to be charged with gNO in theprocess presented herein preferably does not sequester gNO in advance,prior to effecting the process. Hence, according to some embodiments ofthe present invention, the article is untreated with gNO at the time itis first placed in the chamber of the gNO charging device.

Thus, in the context of embodiments of the invention, the article whichis exposed to gaseous nitric oxide-containing environment can be seen asan intact article, whereby the phrase “intact article” is meant todescribe an article that has not been exposed to gNO-containingenvironment.

The process according to embodiments of this aspect of the presentinvention starts by placing an article in a chamber. The article placedin the chamber can therefore be regarded as an intact article.

The chamber can be any tank, canister, vat, barrel, cask, hogshead,drum, case, wrapper, sheath, bag, compartment, vessel, container orreceptacle which can serve as encasement for the article in terms ofsize (internal size) and capacity to contain gases, and particularly gNOcontaining environment. By capacity to contain, it is meant that thechamber is sealed to an extent that allows charging the article, andmechanically fit to sustain both negative and positive pressure. Suchrequirements typically translate to mechanical integrity for maintainingimpermeability to gases, rigidity and/or durability to maintain negativeand positive pressure, and the ability to be fitted with inlets andoutlets without losing containment of gases, while maintaining theintegrity of the article disposed therein.

The chamber forms a part of a charging device, which is designed tocarry out the process presented herein and includes inlets and outlets(a single or a plurality of each); tubes for connecting the inlets andoutlets to the chamber, to external sources, pumps and exhausts andtherebetween; various inlet and/or outlet valves; various optional inletand/or outlet gauges for monitoring inflow and outflow of gases; andvarious optional absorbers and scavengers of undesirable contaminants,as well as gauges and indicators for monitoring the environment withinthe chamber at various steps of the process, as is further detailedhereinunder.

The process involves placing an article of interest (an article to beimpregnated) into the chamber with the intention of loading the articlewith gNO.

The article disposed within the chamber can be anyarticle-of-manufacture, any part of an article-of-manufacture or a stockor raw material for manufacturing an article-of-manufacture ormanufacturing a part of an article-of-manufacture. For example, in casesthe article is a medical device having various surfaces, coating,appendages and tubing, the process may be carried out by placing theentire medical device in the chamber, placing some of the parts of themedical device, such as tubing or surface-forming parts of the medicaldevice in the chamber, or placing in the chamber the pre-processed,pre-shaped, pre-formed or pre-cut raw materials constituting parts ofthe medical device.

Once the article in disposed within the chamber, the process involvesgenerating a reduced pressure in the chamber. The term “reducedpressure”, is used synonymously with the terms “negative pressure”“under pressure” and/or “vacuum”. Evacuating the chamber from theambient environment substantially evacuates (removes) gaseous substancesand/or volatile substances found in the chamber as well as gaseoussubstances and/or volatile substances found in the article, at least tothe extent of parts of the article which are exposed to the negativepressure.

In the context of embodiments of the invention, the term “gaseous”refers to a state of a substance being a gas under certain conditions ofpressure and temperature. For example, the melting point of nitric oxideat atmospheric pressure is −164° C. and the boiling point of nitricoxide is −152° C., hence nitric oxide is a gaseous substance at ambientconditions of pressure and temperature (i.e., room temperature). Oxygen,nitrogen and CO₂, present in an ambient atmosphere, are also gaseoussubstances. Humidity and moisture forming-water is essentially a mixtureof vapor (gaseous water) and liquid water at ambient conditions ofpressure and temperature, however under reduced pressure the equilibriumof gas-liquid of water would essentially shift towards the gaseousstate. Hence, reducing the pressure in the chamber, according to someembodiments of the present invention, facilitates the removal ofmoisture and humidity from the chamber prior to introducing gNO therein.

Generating a reduced pressure in the chamber can be effected byconnecting any inlet or outlet of the charging device to an externalsource of reduced pressure, such as a vacuum pump, or a vacuumreservoir. A vacuum reservoir can be in the form of, for example, acontainer which has been evacuated from its content to posses a volumeunder reduced pressure which is substantially larger than the volume ofthe chamber, thereby being capable of taking-in (sucking in) at least apart of the ambient atmosphere of the chamber.

In the context of embodiments of the invention, the level of the reducedpressure which is reached in the chamber can be at any level of vacuumwhich is reasonably attainable in the charging device resented herein.According to some embodiments of the present invention, the depth of thevacuum can range from low vacuum levels (about 100 kPa to 3 kPa) to highvacuum levels (about 100 mPa to 100 nPa). In the context of embodimentsof the invention, vacuum levels can be expressed as a negative value,namely by the value representing the difference in pressure relative toatmospheric pressure (which is 760 Ton, 101.23 kPa, 14.7 psi or 1atmosphere). Hence, according to some embodiments of the presentinvention, the reduced/negative pressure attained in the chamber canrange from about −50 psi to −0.5 psi. The unit “psi” is used in thecontext of some embodiments of the present invention, to denote apressure difference, typically as recorded by a gage or device, from areference pressure, typically atmospheric pressure. “0 psi” thereforedenotes atmospheric pressure (e.g., ambient pressure), whereby “−X psi”is used to express negative pressure (under pressure).

In some embodiments of this aspect of the present invention, generatingthe reduced pressure in the chamber is effected for a time period of atleast 1 minute, or for a time period that ranges from 1 minute to 60minutes, or from 1 minute to 30 minutes, or from 1 minute to 20 minutes,or from 1 minute to 15 minutes, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes, or for 30, 40, 50 or60 minutes. During this period, the article is exposed to reducedpressure and in essence undergoes a purging process during which gasesand volatile substances are substantially removed therefrom. Thegeneration of reduced pressure in the chamber can thus be regarded as adegassing process or as a gas-evacuation process.

It is noted herein that water and gNO tend to interact such that thecapacity of a moist article to sequester gNO is reduced. It is furthernoted herein that gNO is a relatively hydrophobic substance and hencethe presence of water (moisture of humidity) can adversely interferewith sequestering gNO. Hence, it is desirable to reduce the humidity inthe article prior to exposing the article to gNO-containing environment.

Oxygen and gNO also interact to produce reactive species such asnitrogen dioxide (nitrate), according to the reaction 2NO+O₂→2 NO₂,which is a toxic brownish gas. Water, oxygen and gNO react to producenitrite according to the reaction 4NO+O₂+2H₂O→4HNO₂. Nitrates andnitrites are known to participate is various reactions in vivo, in whichtoxic reactive oxygen species are formed. These reactions and subsequentreactions involving products of these reactions are commonly referred toherein as gNO degradation, and the process presented herein attempts tominimize this gNO degradation, by minimizing the presence of reactivespecies other than gNO.

Nitrates, nitrites, and any other species that are formed directly orindirectly by a reaction of nitric oxide with oxygen and/or water orhumidity are referred to herein as “reactive species other than nitricoxide” or simply as “reactive species” and are meant to include nitrogenand/or oxygen-containing reactive species.

In some embodiments, generating reduced pressure in the chamber iseffected so as to reduce the humidity level in the article/chamber by atleast 50% of its original (ambient) level. According to some embodimentsof the present invention, generating reduced pressure is effected so asto effect a decrease the humidity level in the article by more than 50%,more than 60%, 70%, 80%, 90% and up to 100% reduction in humidity, whichis essentially desiccating the article to a level of humidity of almostzero or essentially zero humidity. Preferably, the amount of humidity(relative humidity) in the ambient environment prior to introducing gNOinto the chamber is reduced such that the relative humidity in thechamber prior and during the introduction of the gNO-containingenvironment can be from about 0% to about 25%, or any amounttherebetween, for example, about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% or 24%.

In the context of oxygen found in the article and/or the interior of thechamber, generating reduced pressure in the chamber is effected so as toeffect reduction of oxygen level in the article/chamber by at least 50%of its original (ambient) level. According to some embodiments of thepresent invention, generating reduced pressure may decrease the oxygenlevel in the article by more than 50%, more than 60%, 70%, 80%, 90% andup to 100% reduction in oxygen level, which renders the articleessentially devoid of oxygen.

According to some embodiments of the present invention, generatingreduced pressure in the chamber is effected so as to effect reduction ofboth oxygen and humidity levels to below 50%, or below 40%, 30%, 20%,10% and down to essentially 0% of the ambient levels of each of oxygenand humidity before the exposure to gNO-containing environment.

In some embodiments, once the article in placed within the chamber, thechamber is sealed so as to allow the application of reduced pressuretherein for any desired length of time. After sealing and generating areduced pressure in the sealed chamber, the ambient environment in thechamber can be replaced with a gNO-containing environment as describedhereinbelow.

Once most of the ambient environment, which may contain undesired levelsof water and oxygen which may react with gNO, has been substantiallyremoved by generating reduced pressure from within the chamber and thearticle, the chamber is filled with a replacement environment whichcomprises gaseous nitric oxide. In some embodiments, the gNO-containingenvironment is an ambient environment comprising nitric oxide. Thephrase “ambient environment comprising nitric oxide” and the phrase“gaseous nitric-oxide containing environment” refer equally to pure gNOor to any mixture of gNO and a carrier gas. A carrier gas can be anyinert or otherwise biologically and chemically compatible gas such as,but not limited to, helium, argon, nitrogen gas and any combinationthereof.

It is noted herein that filling the chamber with a gNO-containingenvironment can be performed by allowing a gNO-containing environment toflow into the chamber, namely, by simply connecting the chamber, inwhich reduced pressure was generated, to a source of the gNO-containingenvironment. This gaseous environment will flow into the chamber due topressure differences. In some embodiments, filling the chamber with agNO-containing environment can be performed by pushing thegNO-containing environment into the chamber at an elevated pressure, orallowing the gNO-containing environment to sucked into the chamber byforce of the vacuum present therein.

Filling the chamber substantially charges the article with gNO. ThegNO-containing environment which is introduced into the chamber containsa predetermined concentration of gNO which is capable of exerting thedesired effect of NO on the article, as discussed herein. Thepre-determined concentration of gNO in the provided gNO-containingenvironment may range from about 0.05% to about 10%, or any amounttherebetween, for example about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9% or 10% of the total amount of the gNO-containingenvironment, with the remaining environment being the carrier gas. Theamount of gNO can alternately be expressed in parts per million (ppm),wherein ppm and percent values are readily interconverted; for example100 ppm is 0.01%, 1,000 ppm is 0.1%, 10,000 ppm is 1%, and 100,000 ppmis 10%. According to some embodiments of the invention, theconcentration of gNO in the gNO-containing environment ranges from 160ppm to 50,000 ppm. The gNO concentration within the gNO-containingenvironment can be determined by methods well known and widely practicedto those skilled in the art.

The gNO-containing environment may be provided (maintained, supplied asa continuous flow or as a single disbursement) for a pre-determinedamount of time that ranges from about 1 minute to about 24 hours, namelyfrom about 1 minute to about 24 hours, or any period therebetween, forexample about 1, 2, 5, 10, 15, 20, 30, 40, 50 or 60 minutes, or about 1,2, 4, 6, 8, 10, 12, 16, 18 or 24 hours or higher, and any period of timetherebetween.

A continuous flow of gNO-containing environment can be effected at aflow rate that ranges from about 1 cubic centimeter or milliliter perminute (cc/min) to about 2,000 cc/min or any rate therebetween, forexample 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750,1,000 or 2,000 cc/min, or any rate therebetween. According to someembodiments, the flow rate ranges from about 250 cc/minute to 1,700cc/min.

The ability to control the flow rate, concentration of gNO in thegNO-containing environment and the duration of exposure serve as threefinely tuned means to adjust the amount of gNO sequestered in thearticle. For example, a higher concentration of gNO in the carrier gaswill result in an article sequestering a higher amount of gNO.Maintaining the article exposed to the gNO-containing environment for alonger time will result in an article sequestering a higher amount ofgNO. Similarly, increasing the flow rate of the gNO-containingenvironment through the chamber corresponds to increasing the amount ofgNO which the article is exposed to, resulting in an articlesequestering a higher amount of gNO. In some cases the article cannotsustain high vacuum levels to long or any duration of time, and in thesecases controlling the amount of sequestered gNO will be achieved byconcentration and flow rate of the gNO-containing environment.

It is noted herein that the goal level of sequestered gNO in the articlecan be achieved in more than one way, using all possible variablespresented herein (such as, for example, gNO concentration, duration ofexposure, flow of gNO-containing environment, degassing by vacuum,desiccation and oxygen purging).

It is, however, noted herein that the mode by which the desired amountof sequestered gNO is achieved in the article depends on the nature andthe composition of the article. Articles of a higher volume and/or bulkyarticles and/or articles made of a material with lower affinity to NOand/or articles that require particularly high level of sequestered NO(due to the intended use thereof) typically require higher loading timeand/or higher duration and/or extent of reduced pressure and/or higherflowing rate of gaseous NO.

Undershooting the optimal parameters of the aforementioned variableswill result in an article with sub-effective level of sequestered gNO,and overshooting may damage the article or some of its components and/orthe chamber.

According to some embodiments of the present invention, theconcentration of gNO in the gNO-containing environment ranges from 150ppm to 60,000 ppm.

According to some embodiments of the present invention, theconcentration of gNO in the gNO-containing environment ranges from 800ppm to 20,000 ppm; the period of time of exposure to the environment inthe chamber ranges from 1 to 600 minutes or overnight (10-24 hours);and/or the flow rate at which the environment is introduced into thechamber ranges from 0.5 liter/minute to 5 liter/minute.

During the step of dosing (filling the chamber and maintaining theaforementioned amount of gNO in the chamber for the aforementionedperiod of time), the article or at least a portion thereof, maysequester gNO at a range of 1 ppm to about 20,000 ppm. Hence, the resultof the process presented herein is essentially an article (releasably)sequestering gNO in at least a portion thereof, as discussedhereinbelow. In some embodiments, the process results in an articlesequestering from 1 ppm to 200 ppm gNO per cm³ of the article. In someembodiments, the process results in an article sequestering from 1 ppmto 200 ppm gNO as the total amount of gNO. However, higher amounts ofgNO are also contemplated, as well as any value between the 1 ppm and20,000 ppm.

In general, it is noted herein that the amount of gNO sequestered in anarticle impregnated upon generating a reduced pressure is higher by atleast 5%, 10%, 20%, 30%, 40% and even 50% compared to the gNOsequestered in an article without prior generation of reduced pressure.

Once exposure to gNO-containing environment is completed, the process,according to some embodiments of the present invention, involvesevacuating the residual gNO-containing environment from the chamber, inpreparation to opening the chamber and retrieval of the treated articlefrom the charging device. Such a step can be effected by purging thechamber by flowing a carrier gas or ambient atmosphere into the chamber,by pumping the gNO-containing environment from the chamber with a vacuumpump or by a combination of pumping and purging.

Depending on the material(s) making the article, the amount of gNO inthe environment, the duration of exposure to the environment duringdosing and on various other factors as described hereinabove, theresulting gNO-sequestering article or at least portions thereof, aredosed with a predetermined concentration of gNO which is sufficient tosustain release of an effective amount of gNO (e.g., for maintainingsterility) at storage conditions for a time period ranging from about 1day to about 5 years, or from about 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3weeks, 4 weeks, about 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 42, 48, 54 orabout 60 months, or any period therebetween.

By “storage conditions”, it is meant that the article is kept underconditions which maximize the length of time that gNO can be releasedtherefrom at an effective amount once it is removed from storageconditions. The term “storage conditions” encompasses any form ofmaintaining a gNO-containing environment in the immediate vicinity ofthe article, either in the charging device, the sealed chamber or in anon-gas permeable container or package, as these are discussedhereinbelow. Storage conditions also include maintaining an environmentwhich is low in oxygen, moisture, heat or other factors which may reducethe levels of gNO sequestered in the article.

As mentioned hereinabove, the amount of sequestered gNO depends, amongother factors, on the size, volume, surface area and composition of thearticle.

As known in the art, different substances have different solubility indifferent media, and this general principle holds also in the case ofthe inherent capacity of a substance to sequester gNO at any givenconditions. Articles can be selected to be made from materials having ahigh intrinsic capacity to sequester gNO, or alternatively, one canselect parts of the article which are made from materials having a highintrinsic capacity to sequester gNO, to be assembled into a completearticle.

Materials having a high intrinsic capacity to sequester gNO, accordingto some embodiments of the present invention, include polymers,co-polymers and resins of various chemical compositions and othermechanical and morphological properties.

Articles are made primarily from materials which are most suitable toserve the intended use of the article, such as, for example, medicaldevices, which are generally made from polymeric materials that arecompatible with the internal or external surfaces of tissues and organsof a living subject (biocompatible).

In some embodiments, the article includes at least a portion of asurface configured to sequester nitric oxide, preferably a surfaceconfigured to sequester at least 1 ppm nitric oxide per cm³, andoptionally, a surface configured to sequester at least 200 ppm nitricoxide per cm³, depending on the intended use of the article.

In some embodiments, at least a portion of the article comprises aplurality of voids for accepting and sequestering nitric oxide. Articlesthat comprise voids may be comprised of polymeric materials that have acertain degree of porosity due to e.g., chemical composition andmanufacturing parameters of the polymeric material (e.g., cross-linkedpolymers) and/or due to physical parameters such as articles made ofcondensed layers or fibrous structures of a (e.g., polymeric) materialor a plurality of materials.

In general, articles that have at least a portion thereof which isgas-permeable are suitable for use in the context of these embodimentsof the present invention.

In the context of embodiments of the present invention, polymericmaterials, polymers, co-polymers and resins which are both suitable toserve the intended use of the article they comprise, and have asufficient intrinsic capacity to sequester gNO are commonly referred toherein as “suitable polymeric materials”.

Examples of suitable polymeric materials for forming polymericmaterial-based medical devices include resins which may exhibitmicroporous structure, and which are generally gas-permeable, and areable to sequester gNO. Upon contact with moisture exemplified by water,saline or other irrigation fluids, and/or bodily fluids such as urine,blood, mucus, and the like, the sequestered gNO can be releasedcontrollably from these compatible polymers over an extended period oftime, thereby creating a localized microbial-free environmentsurrounding the medical device, or a portion thereof, thus rendering themedical device self-sterilizing.

Since any gNO-permeable material wrapping or encasing the article wouldundergo gNO dosing at the same time as the article, or at least allowgNO to pass-through to impregnate the article therein, the term“article”, as used herein, is meant to encompass an entire article andany appendage or supplemental part attached or connected thereto,including, without limitation, any gNO-permeable wrapping, coatingand/or encasement material completely engulfing or partially encasingthe article, and in particular any gNO-permeable wrapping or encasementwhich can also sequester gNO.

Chemical composition is one of the characteristics that influence amaterial's intrinsic capacity to sequester gNO. For example, suitablepolymeric materials are permeable to gNO by virtue of their composition,degree of crosslinking, crystallinity and the likes. In addition, sincegNO is substantially non-polar, it can diffuse more readily intonon-polar media, such as, without limitation, hydrophobic polymers andin general, polymers composed of non-polar constituents.

Hydrophobic polymers have a high intrinsic capacity to sequester higheramounts of gNO and further to sequester gNO for extended periods oftime, due to higher affinity of gNO to hydrophobic substances. Hence,hydrophobic polymers are suitable in applications where prolongedrelease of gNO is beneficial.

It is noted herein that suitable polymeric materials in the context ofembodiments of the present invention are not limited to hydrophobicpolymers, and hydrophilic or amphiphilic polymeric material can also beimpregnated with gNO effectively, according to some embodiments of thepresent invention.

Less hydrophobic and hydrophilic polymers and other substances may stillbe charged with gNO using the aforementioned factors of reducedpressure, gNO concentration in the environment, time of exposure to theenvironment and filled voids occurring in the impregnated substance.

It is noted that less hydrophobic, amphiphilic and hydrophilic polymerstypically require exposure to gNO-containing environment for longer timeperiods and/or at higher flow rates and/or upon generating reducedpressure for longer time periods.

In general, a hydrophobic material is characterized by an oil-in-waterpartition coefficient (LogP) greater than 0, greater than 1, greaterthan 2, greater than 5 or higher.

Non-limiting examples of suitable polymeric materials useful in makinggNO-charged articles, such as for example medical devices, includesilicone, polyacetal, polyurethane, polyester, polytetrafluoroethylene,polyethylene, polymethylmethacrylate, polyhydroxyethyl methacrylate,polyvinyl alcohol, polypropylene, polymethylpentene, polyetherketone,polyphenylene oxide, polyvinyl chloride, polycarbonate, polysulfone,acrylonitrile-butadiene-styrene, polyetherimide. polyvinylidenefluoride, copolymers, polysiloxane, fluorinated polysiloxane,polyanhydride, ethylene vinyl acetate, methacrylic acid, ethylene oxide,propylene oxide, polystyrene, ethylene-propylene rubber,fluoroelastomer, silastic elastomers, polyethylene tetrephthalate,colloidion, carbothane, nylon, and combinations thereof.

Other examples of suitable polymeric materials include biodegradable andbioresorbable polymers exemplified by polyglycolic acid (PGA),polylactic acid (PLA), poly(L-lactide), poly(DL-lactide),poly(DL-lactide-co-glycolide), polycaprolactone, poly(e-caprolactone),polydioxanone, polyparadioxanone, polytrimethylene carbonate,polyoxalate, a polyanhydride, a poly(phosphoester), or copolymers and/orcombinations thereof, as well as biologically derived materials such asrayon, cotton, catgut, collagen, silk, elastin, chitin, chitosan,cellulose, cellulosic polymers, coral, hyaluronic acid, bone,hydroxyapatite, bioabsorbable calcium phosphate and any combinationsthereof.

Additionally, suitable polymeric materials can be used as coating on anarticle such as a medical device, or serve as encasement of the medicaldevice, whereby the coating or encasement provides gNO-sequesteringcapacity to the medical device. For example, an implantable pump whichforms a part of an implantable drug delivery system can be encasedwithin one or more layers comprising one or more of the suitablepolymeric materials exemplified herein.

Mechanical and morphological properties also influence a material'sintrinsic capacity to sequester gNO. For example, suitable polymericmaterials can be prepared such that they possess a porousmicrostructure, ranging from solid compositions having microscopic voidsdispersed sparsely therein to low density foams having more voids thansubstance. Hence, suitable polymeric materials may range widely on thescale of degree of porosity. In the context of embodiments of thepresent invention, polymers and resins having a porous microstructure toany degree of porosity, referred to herein as “porous substances”, cansequester gNO by virtue of having gNO fill the voids during the chargingprocess. It is noted herein that such voids act as receptacles for gNO,and hence in the context of a substance having voids the term“sequestering” is regarded also as “accepting” or “containing” gNO insaid voids.

The texture of the polymeric material can range from rigid to soft, andbe elastic, plastic, pliant and resilient. The degree of rigidity,elasticity, plasticity, pliability or resilience of suitable polymericmaterials can vary with the specific design and application of anarticle, such as a medical device. The choice of characteristics for aselected medical device will be apparent to and within the ability of aone of ordinary skill in the art. As the aforementioned propertiescorrelate to certain chemical properties such as crosslinking, suchmechanical properties may also correlate with the capacity of apolymeric material to sequester or accept gNO.

According to some embodiments of the present invention, the articleincludes at least a portion of a surface configured to sequester gNO.

As the portion of the article can be defined by its volume, the amountof gNO sequestered in that portion can be quantified by ppm of gNO pervolume unit. Hence, according to some embodiments of the presentinvention, the article includes at least a portion of a surfaceconfigured to sequester at least 1 ppm nitric oxide per cm³.

FIG. 27 presents an illustration of charging device 300, wherein article301 further includes at least a portion thereof made from a suitablepolymeric material, represented by surface 372, and configured tosequester gNO readily and effectively.

As discussed hereinabove, the methodology described herein is highlybeneficial for preparing a medical device having gNO sequesteredtherewithin.

Thus, in some embodiments, the article to be charged with gNO is amedical device. Non-limiting examples of medical devices which can beimpregnated with gNO beneficially, include catheters, tubing,endotracheal tubing, tampons, prosthetic devices, medical implants,artificial joints, artificial valves, needles, intravenous accessdevices, cannula, stents, biliary stents, cardiovascular stents, cardiacsurgery devices, nephrostomy tubes, vascular grafts, infusion pumps,adhesive patches, sutures, fabrics, meshes, polymeric surgical tools orinstruments, intubation devices, orthopedic surgery devices, pacemakers,endoscope components, dental surgery devices, veterinary surgerydevices, bone scaffolds, hemodialysis tubing or equipment, bloodexchanging and transfusion devices, implantable prostheses, bandages,ophthalmic devices and breast implants.

According to some embodiments of the invention, the medical device is animplantable medical device, including medical devices that are implantedtransiently or permanently. Examples include an indwelling catheter oran intubation device such as a tracheal tube. Non-limiting examples ofindwelling catheters include urinary catheters, central venouscatheters, biliary vascular catheters, pulmonary artery catheters,peripheral venous catheters, arterial lines, central venous catheters,peritoneal catheters, epidural catheters and central nervous systemcatheters.

As discussed hereinabove, while CAB is one of the major causes ofnosocomial infections, sterilization of urinary catheters is highlydesired. Preparing urinary catheters the releasably sequester gNO thusachieves this goal (amongst other goals that are met by the describedmethodology).

In some embodiments, the medical device is such that is made of any ofthe suitable polymeric materials described hereinabove (e.g., at least aportion of the medical device comprises a hydrophobic polymeric materialwith an intrinsic capacity to sequester gNO).

According to some embodiments of the invention, the medical device is atampon. As discussed hereinabove, gNO has been shown to bebacteriostatic and bactericidal, and has been shown herein to beeffective in eradicating microorganisms causing bacterial vaginitis.Apart from self-sterilization, gNO-charged tampons can serve also as atherapeutic medical device by delivering a medication (gNO) to aninfected bodily site (vagina).

Since tampons are made essentially from fibers of less hydrophobicand/or even hydrophilic polymers and other substances, impregnatingtampons with gNO can advantageously be effected under reduced pressureand/or using extended gNO exposure times and/or at higher gNOconcentrations compared to an article made from hydrophobic polymers.However, as tampons are designed for absorbing liquids, theirmicrostructure is essentially rich in voids and crevices which can becharged and filled with gNO, sequester and contain gNO, and effectivelyand controllably release sequestered gNO therefrom. Additional featuresrelated to gNO-impregnated tampons are discussed in further detailhereinbelow.

As discussed hereinabove, the article used in the methodology describedin these embodiments of present invention encompasses bare (unwrapped)articles, coated articles, and wrapped articles, as long as the coatingand/or wrapping does not impair the process of gNO loading to an extentthat is not mitigated by means within the process (namely byprocess-controllable factors that can increase gNO uptake).

This definition therefore makes the distinction between gas-permeableand non-gas permeable (gas-impermeable) packaging and wrapping, sincethe latter impairs the gNO charging process when the package is sealedand gNO cannot defuse therethrough. An unsealed gas-impermeable packagehaving an article housed therein does not impair gNO penetration to thearticle and hence is regarded as an open or unsealed package and not asan effective gNO barrier.

According to some embodiments of the present invention, the processpresented herein can be effected with a packaged article as well as witha bare (unpackaged) article.

In some embodiments, the package is a gas-permeable package.

The term “gas-permeable” refers to a chemical attribute of a material,which allows gas molecules to pass therethrough by flux, conveyance,diffusion or transportation. “Gas-permeability” also refers to amechanical attribute of an article made from any material in the sensethat the material can be shaped, designed, manufactured and processed tobe impermeable, permeable and anywhere between the extremes(semi-permeable). A material can be made porous or caulked, shaped thinor thick, processes glassy or rough and the likes, all of which areexamples of mechanical propertied that affect gas-permeability. In thecontext of embodiments of the present invention, semi-permeability inthe sense of rate of permeation is regarded as gas-permeable, andsemi-permeability in the sense of selectivity towards one gas species toanother is regarded as gas-permeable if it can permeate gNO, andgas-impermeable if it does not allow gNO to permeate therethrough.

Non-limiting examples of gas-permeable materials include low densitypolyethylene (LDPE), high density polyethylene (HDPE), medical gradepaper, polycarbonate (PC), polyester, polyvinyl chloride (PVC),polyvinylidene chloride, perfluoroalkoxy (PFA), acrylobutylstyrene,polypropylene (PP) polytetrafluoroethylene (PTFE), polyacrylate,acrylic, polycarbonate, polyacrylonitrile-butadiene-styrene,polymethylpentene (PMP), polyacetal, polystyrene (PS) or the like.

Hence, according to some embodiments of the present invention, thearticle can be housed in (e.g., enclosed by) a gas-permeable packagewhen disposed in the chamber during the process of generating a reducedpressure and charging the article with gNO; wherein the presence of thegas-permeable package may or may not affect one or more parameters ofthe process. The end result of the process would be a gNO-chargedarticle that includes a gNO-charged gas-permeable package.

In some embodiments, the process described herein is used for providingan article as described herein, with or without a gas-permeable package,within a non-gas permeable enclosure, in order to maintain thegNO-containing environment within the package for prolonged time and/orunder various conditions.

The term “non-gas permeable” or the equivalent “gas-impermeable”, asused herein, refers to an attribute of a substance which is capable ofpreventing the passage of gas molecule therethrough by flux, conveyance,diffusion or transportation. In some embodiments of the invention, theseterms refer to impermeability of gNO, however, this term is also used toindicate impermeability of the non-gas permeable to other gases such aswater vapors and oxygen. Without being bound by a particular theory, itis assumed that since gNO has a lower diffusion cross-section then watervapors or oxygen, a gNO-impermeable substance forming the non-gaspermeable container or package will also be impermeable to water vaporsand oxygen, and that a substance that is impermeable to water vapors andoxygen may still be permeable to gNO.

Examples of materials suitable for making gas-impermeable packaginginclude, without limitation, glass, glassy ceramics, metals, metallicfoils, metallic-plastic composite foils and the likes and a combinationthereof as composites or as parts in a complete gas-impermeable package.The gas-impermeable material for packaging can also comprise more thanone layer of a polymer, a metal, a resin or a plastic, and in someexamples the packaging can comprise a plastic-backed metallic foil, suchas used in many air-tight packaging. A gas-permeable material can becombined with a gas-impermeable material to form a compositegas-impermeable package.

In the context of embodiments of the present invention, agas-impermeable package is also referred to interchangeably as a non-gaspermeable enclosure or gas-impermeable enclosure.

According to some embodiments of the present invention, the article ishoused in an unclosed (unsealed, open) sealable gas-impermeable package(a non-gas permeable enclosure that can be closed and sealed) whendisposed in the chamber and during the generation of reduced pressureand charging it with gNO. According to such embodiments, the process iscarried out essentially as described hereinabove, and at the end of theprocess the non-gas permeable enclosure is closed and sealed so as toconstitute an intact and complete gNO barrier with respect to the gNO inthe gNO-containing environment and the gNO-sequestering article enclosedtherein, insulating the article from ambient environment.

The sealable gas-impermeable package for insulating a gNO-sequesteringarticle enclosed therein can further include a desiccant, a humidityindicator and a gNO indicator, as these are described herein, formonitoring the gNO-charging process and to indicate post-sealingintegrity of the package and post-sealing level of gNO enclosed thereinafter manufacturing. These optional attachments may be placed inside thepackage and behind a transparent portion of the package so as to allow avisual signal generated therein to be detected without opening thesealed package. Alternatively the indicators may be designed to form apart of the gas-impermeable package such that the inner side is incommunication with the inner environment and can interact therewithwhile the outer side of the indicator maintains gas-impermeabilitytowards the outside ambient environment.

Additional details of a process involving preparing an article packagedin a non gas-permeable enclosure are provided hereinafter.

Utilizing the process described herein, articles having gNO sequesteredtherein are provided. Such articles are advantageously characterized ascomprising at least 1 ppm per cm³ nitric oxide sequestered therewithin,optionally at least 200 ppm per cm³ nitric oxide sequestered therewithin, and further optionally from 1 ppm to 20,000 ppm per cm³.

In some embodiments, the gaseous nitric oxide sequestered in the articleis releasable is an aqueous solution during a time period that rangesfrom 1 hour to 1 month.

The rate of release of NO depends on a variety of parameters, including,for example, the affinity of NO to the material composing the article(the material in which gNO is sequestered), the amount of sequesteredgNO, the position of gNO within the article (being an external orinternal surface), as well as the conditions at which gNO is released.

As discussed herein, while considering the nature of the article (itsstructure and chemical composition, and more particularly, but notexclusively, its affinity to NO, its size, volume and bulkiness, etc.)and its intended use, the amount of gNO charged into the article can bepre-determined, so as to impart a desired release rate of gaseous NOtherefrom.

For example, urine catheters are typically transiently implanted inpatients for a period of from a few hours to a few days. Such articleswould therefore be loaded by a process that utilizes conditions for highloading of NO (e.g., longer duration of exposure to NO-containingenvironment).

Tracheal tubes are typically utilized for a few hours, yet require highload of gNO since are typically used during surgery or other emergencycases.

Tampons are intended to be replaced every few hours.

The amount of gNO sequestered in the articles described herein can bereadily measured by measuring the amount of nitrites and/or nitrates, asgNO degradation products, in an aqueous solution in which thegNO-sequestering article is placed. Determining the amount of nitritesand nitrates is exemplified in Examples section that follows. Othermethods for quantitatively determining nitrites and nitrates will berecognized by a person skilled in the art and are also contemplatedherein.

The article disclosed herein is further characterized as beingsubstantially devoid of humidity and/or oxygen.

In some embodiments, the article described herein is characterized by nomore than 25% of humidity, no more than 20% humidity, no more than 15%humidity, no more than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%,0.01%, 0.005%, 0.001% humidity, and preferably by 0% humidity. The “%”represents weight percentages from the gaseous environment within thearticle.

Alternatively, or in addition, in some embodiments, the articledescribed herein is characterized by no more than 20% of oxygen, no morethan 15% oxygen, no more than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%,0.05%, 0.01%, 0.005%, 0.001% oxygen, and preferably by 0% oxygen. The“%” represents weight percentages from the gaseous environment withinthe article.

In some embodiments, the article comprises no more than 1 ppm of oxygenand/or humidity.

In some embodiments, the article comprises no more than 1 ppb of oxygenand/or humidity.

In some embodiments, an amount of reactive species other than nitricoxide, as defined herein, in the article is lower than 1 ppm per cm³ ofthe article, preferably lower than 1 ppb (part per billion) per cm³ ofthe article.

In some embodiments, an amount of reactive species other than nitricoxide, as defined herein, in the article, is lower than 1 ppm of theentire article, preferably lower than 1 ppb (part per billion) of theentire article.

Depending on the process particulars and the article loaded with gNO,the article may further comprise an enclosure (e.g., a non gas-permeableenclosure).

In some embodiments, the enclosure comprises a gaseous nitricoxide-containing environment.

In some embodiments, the environment is an ambient environment (e.g.,has an ambient pressure).

In some embodiments, the enclosure contains a gaseous NO-containingenvironment with a concentration of NO therein that is sufficient tosterilize the interior of the package and/or the article, as is furtherdiscussed hereinunder.

The article and/or enclosure can further comprise a desiccant disposedwithin the enclosure, and/or a nitric oxide indicator disposed withinthe enclosure, as is further discussed in detail hereinunder.

Hence, according to as aspect of some embodiments of the presentinvention there is provided an article having sequestered therewithin atleast 1 ppm gaseous nitric oxide, which is advantageously characterizedas comprising less than 1 ppm, or less than 1 ppb, per cm³ of thearticle, reactive oxygen-containing and/or nitrogen-containing speciesother than nitric oxide.

The article can be further characterized by any of the featuresdescribed hereinabove.

In some embodiments, the article is a medical device, as describedherein.

In some embodiments, the article is a tampon.

The term “tampon”, as used herein, refers to a medical device in theform of a plug made from a mass of absorbent materials which is insertedinto a wound or a body site to absorb exuded fluids, such as blood. Oneof the most common types of tampons in daily household use is designedto be inserted into the vagina during menstruation to absorb the flow ofmenstrual fluid. Such tampons are regarded officially as medical devicesin many courtiers around the world, and according to the United StatesFood and Drug Administration, tampons are a Class II medical device.

In some embodiments, the term “tampon” describes tampons designed to beinserted into the vagina during menstruation to absorb the flow ofmenstrual fluid. The tampon can be a commercially available tampon ofany type, composition, absorption rate, size and/or blend.Alternatively, one or more of the polymeric materials used for forming atampon have a gaseous NO sequestered therewithin, and the tampon is madefrom these raw materials.

Tampons are typically made from cotton, rayon and blends thereof, andare available in different sizes for various conditions and absorbingrates. Tampons may include an applicator, which is a polymeric tubesheathing the absorbent plug for facilitating its insertion into thevagina.

Both cotton and rayon, comprising the major and absorbing part of atampon, are cellulosic fibers, which are regarded in the context ofembodiments of the present invention as hydrophilic polymers.

As discussed hereinabove, tampons releasably sequestering gNO are highlyadvantageous, particularly in the context of prevention and/or treatmentof vaginal medical conditions such as vaginal infections.

As demonstrated in the Examples section that follows, the presentinventors have successfully prepared tampons having sequestered thereingaseous NO and have shown that such tampons exhibit both anantimicrobial effect and a self-sterilization effect (by e.g.,preventing bacterial growth, biofilm formation and/or bacterial growthwithin a biofilm).

Due to the hydrophilic nature of the material(s) composing tampons,discussed hereinabove, the ability to have gaseous nitric oxidesequestered therewithin is surprising.

Thus, according to an aspect of some embodiments of the invention thereis provided a tampon having sequestered therein gaseous nitric oxide.

According to some embodiments, an amount of the sequestered gaseousnitric oxide ranges from 1 ppm to 200 ppm per cm³ of the tampon.Alternatively, an amount of the sequestered gaseous nitric oxide rangesfrom 1 ppm to 200 ppm of the tampon.

According to some embodiments, an amount of sequestered gaseous nitricoxide is 200 ppm per cm³. Alternatively, an amount of the sequesteredgaseous nitric is 200 ppm of the tampon. Higher load of gaseous nitricoxide is also contemplated (e.g., 300 ppm, 400 ppm, 500 ppm, 1,000 ppmand up to 20,000 ppm).

According to an aspect of some embodiments of the present invention,there is provided a process for preparing a tampon having sequesteredtherein gNO, which is generally effected by exposing the tampon togNO-containing environment.

As described hereinabove in the context of charging a general article ina gNO-charging device, in some embodiments, the process of preparing agNO-sequestering tampon includes exposing the tampon to gNO-containingenvironment by placing the tampon in a chamber and filling the chamberwith the gNO-containing environment, wherein the exposure is subject tovariable exposure time periods and concentration of gNO in thegNO-containing environment for obtaining a predetermined gNO-load in thegNO-charged tampon.

According to some embodiments of the present invention, the process ofpreparing a gNO-sequestering tampon may further include, subsequent toplacing the tampon in the chamber, sealing the chamber and generating areduced pressure (vacuum) in the chamber, prior to filling the chamberwith the gNO-containing environment.

As described hereinabove, the depth of the vacuum and the duration ofthe time of maintaining the chamber under reduced pressure serve asadditional means to control the amount of gNO sequestered in the tampon.

Optionally, the process of preparing a gNO-sequestering tampon iseffected without generating reduced pressure. Such a process maycomprise, prior to filling the chamber with NO, reducing the humiditywithin the tampon and/or within the chamber.

Since tampons comprise hydrophilic polymers, they require longerexposure to higher concentrations of gNO and optionally more negativepressure applied prior to exposure to gNO, compared to articles ofsimilar mass and shape comprising hydrophobic polymers. However, theprinciples of the process for impregnating tampons with gNO follow thesame guidelines as the principles of impregnating other packaged orunpackaged articles with gNO, as described in detail herein.

When subjected to exposure to gNO-containing environment, the tampon canbe either wrapped or unwrapped.

Apart of optionally being individually wrapped in a gas-permeablewrapping material, tampons may also include an applicator which can bemade from hydrophobic polymers as well as other polymers, and can beregarded as a “gNO-sequestering surface” or a “surface”, as the term isdefined hereinabove, and serves as an additional gNO-sequesteringcomponent in the gNO-sequestering tampon. In the context of embodimentsof the invention, the term “tampon” also encompasses tampons sheathed inan applicator.

As described hereinabove, tampons can be charged with gNO as baretampons or packaged tampons wrapped with a gas-permeable package. Bothforms of tampons can also be charged with gNO while housed in agas-impermeable enclosure, as described hereinabove for charging ageneral article with gNO. The end result of such a process can be eithera gNO-sequestering tampon, a gNO-sequestering tampon packed in agas-permeable package, and/or a wrapped or bare gNO-sequestering tamponpackaged in a gas-impermeable packaging material, either individually oras a plurality of gNO-sequestering tampons.

The gNO-sequestering tampon is self-sterilizing, which adds a notablebenefit for this medical device for insertion into a bodily site,however, a gNO-sequestering tampon may also serve for medicinal therapyof various illnesses associated with the vagina. Hence, according to anaspect of embodiments of the present invention, there is provided agNO-sequestering tampon which is identified for use in treating orpreventing (or preventing the recurrence of) a vaginal medicalcondition.

Accordingly, there is provided a method of treating a vaginal medicalcondition, the method being effected by placing in the vagina of thesubject the gNO-sequestering tampon as presented herein.

Such a method comprises intravaginal administration of gaseous NO.

Accordingly, according to an aspect of some embodiments of the presentinvention there is provided a method of treating a vaginal medicalcondition, which is effected by intravaginal administration of gaseousNO. In some embodiments, intravaginal administration of NO is effectedby a tampon having (releasably) sequestered therein gaseous NO, asdescribed herein.

According to an aspect of some embodiments of the invention there isprovided a tampon having gaseous NO sequestered therein, for use in amethod of treating a vaginal medical condition, by intravaginaladministration of gaseous NO.

According to another aspect of some embodiments of the invention thereis provided a use of a tampon having gaseous NO sequestered therein, forintravaginal administration of gaseous NO, for treating a vaginalmedical condition.

According to another aspect of some embodiments of the invention thereis provided a method of delivering gaseous nitric oxide into a vagina,which is effected by placing in a vagina of a subject in need thereof atampon having gaseous NO sequestered therewithin, as described herein.

Exemplary vaginal medical condition include, without limitation,bacterial vaginitis (BV), toxic shock syndrome (TSS), toxic shock-likesyndrome (TSLS), streptococcal toxic shock syndrome (STSS), vulvovaginalcandidiasis (VVC), chronic or persistent yeast infections (RVVC), asexual dysfunction, a female reproductive system related disorder and apost-surgery vaginal related condition.

According to another aspect of embodiments of the present inventionthere is provided a use of gaseous nitric oxide in the treatment orprevention of a vaginal medical condition. In some embodiments, thegaseous nitric oxide is used in the form of a gNO-sequestering tampon,such as described herein.

As discussed hereinabove, the present inventors have furthercontemplated using a methodology of impregnating gaseous NO forproviding sterilized and self-sterilizing packages of articles.

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a packaged article, wherein thepackaged article comprises a gas-permeable package, the processcomprising exposing a packaged medical device to a gaseous nitricoxide-containing environment, as described herein.

In some embodiments, exposing is effected by placing the packagedarticle in a chamber; and filling the chamber with a nitricoxide-containing environment.

In some embodiments, the process is effected by generating a reducedpressure in the chamber, prior to filling the gNO-containingenvironment, as described in detail hereinabove.

In some embodiments, filling is effected by flowing nitric oxide intothe chamber, as described hereinabove.

In some embodiments, typically in cases where generation of reducedpressure is not effected, the process further comprises, prior tofilling the chamber, absorbing humidity from the package and/or chamber.In some embodiments, absorbing humidity is effected so as to reducehumidity by at least 50%, as described hereinabove.

In some embodiments, filling is effected such that an ambientenvironment (e.g., ambient pressure) is provided within the package.

Such a process results in a packaged article having gaseous nitric oxidesequestered within the article and within the package.

Such a process can be advantageously utilized for sterilizing articlessuch as medical devices which are packaged in a gas-permeable package,without the need to open the sealed package. The package can comprise aplurality of articles, individually packaged articles, and a packagethat comprises individually packaged articles.

In some embodiments, the article is a medical device, as describedherein.

As discussed hereinabove, it is advantageous to provide NO-sequesteringarticles in a non-gas permeable package. The present inventors have thusdevised and successfully prepared and practiced a process of preparingsuch packaged articles.

Thus, according to an aspect of some embodiments of the presentinvention there is provided a process of preparing a packaged article,wherein the packaged article comprises a non-gas permeable enclosure.

The process according to this aspect of embodiments of the invention iseffected by positioning an article (e.g., an intact article) within anon-gas permeable enclosure, to thereby obtain a non gas-permeableenclosure having the article disposed therewithin;

exposing the enclosure to a gaseous nitric oxide-containing environment,so as to introduce into the enclosure the nitric oxide-containingenvironment; and

sealing the enclosure.

The (intact) article placed within the enclosure can be packaged with agas-permeable package or be unpackaged, as described hereinabove.

Exposing the enclosure to a gaseous nitric oxide-containing environmentis effected, according to some embodiments, following the guidelinesdescribed herein, with or without generating reduced pressure in thechamber.

In some embodiments, the nitric oxide-containing environment comprisesat least 0.02% nitric oxide.

Such a process results in an article packaged in a non-gas permeablepackage, wherein the article has gaseous nitric oxide sequesteredtherewithin and/or the package comprises a nitric oxide-containingenvironment.

In some embodiments, the package contains nitric oxide in an amountsufficient to sterilize an interior of the enclosure as well as tosterilize the article.

Such an amount is at least 1 ppm nitric oxide, and range from 1 ppm to200 ppm nitric oxide. In some embodiments, the amount is higher than 200ppm, as described hereinabove.

By “sterilize” it is meant to eliminate substantially all livingmicroorganisms.

“Substantially” encompasses a majority of a population ofmicroorganisms, namely, at least 80%, 85%, 90% 95%, 98%, 995, 99.9% andoptionally 100% of the microorganism population.

According to some embodiments of the present invention, the processpresented herein can be carried out so as to manufacture an articleimpregnated with gNO and encased in a non-gas permeable package asfollows:

placing an article housed in an open and sealable non-gas permeablepackage within a chamber, wherein the package may include any one ormore of the optional humidity indicator, gNO indicator or desiccant;

optionally generating a reduced pressure in the chamber;

filling the chamber with a gNO-containing environment (exposureconditions); and then either

sealing the package under the exposure conditions; and

purging the chamber, opening the chamber and retrieving the sealedpackage encasing the article now sequestering gNO;

or

purging the chamber, opening the chamber and retrieving the open packageencasing the article now sequestering gNO; and

sealing the package under ambient conditions,

thereby preparing the article having gNO sequestered therewithinpackaged in a gas-impermeable enclosure.

Alternatively, the process is carried out essentially as describedhereinabove with an article which is not housed in a non-gas permeablepackage, and the process further includes, subsequent to opening thechamber and retrieving the gNO-charged article, a step of placing thegNO-charged article in a non-gas permeable package and sealing thepackage.

In all the above embodiments a bare article and an article in agas-permeable wrapping, coating and/or packaging is regarded essentiallythe same in the context of the gNO-charging process, since agas-permeable wrapping, coating and/or packaging does not substantiallypreclude or interfere with the process.

Thus, according to an aspect of some embodiments of the presentinvention there is provided a package which comprises a materialconfigured to form an enclosure; an article disposed within theenclosure; and a gaseous nitric oxide-containing environment within theenclosure.

In some embodiments, the package is a non-gas permeable package.

In some embodiments, the enclosure is a sealed enclosure.

In some embodiments, the environment within the enclosure is an ambientenvironment, as described herein.

In some embodiments, the article in the package has gaseous nitric oxidesequestered therein.

The package as described herein can further include desiccants, nitricoxide indicators and other components, as described herein.

The article is preferably a medical device, such as an indwellingcatheter, an intubation device or a tampon, as described herein.

Turning now to the figures, FIGS. 31A-B present schematic illustrationsof a package according to some embodiments of the invention, such as apackage 200, comprising non-gas permeable package 203 configured to beimpermeable to gNO and optionally comprising seal 205 (FIG. 31B),medical device 201 disposed within non-gas permeable package 203,gNO-containing environment 206 engulfing medical device 201 withinnon-gas permeable package 203 and comprising a predeterminedconcentration of gNO capable of sterilizing the interior of non-gaspermeable package 203 and medical device 201, and further optionallycomprising nitric oxide indicator 204 (FIG. 31B), desiccant 202 disposedwithin non-gas permeable package and configured to absorb humidity fromgNO-containing environment 206, and humidity indicator 208 (FIG. 31B)configured to indicate moisture saturation of the desiccant 202.

According to an aspect of some embodiments of the invention, there isprovided charging device (or system), which comprises:

a chamber comprising an inlet for receiving a gaseous nitric-oxidecontaining environment and an outlet for releasing the gaseousnitric-oxide containing environment; and

a article, as described herein, disposed within the chamber.

Such a chamber can be utilized for practicing any of the processesdescribed herein.

The chamber may be coated from within with a protective surface coatingsuitable for preventing gNO from reacting with the chamber's material.Such protective coating may include, as non-limiting examples, glass,chromium, stainless steel and other gNO-resistant substances andmixtures thereof.

The device may further comprise a nitric oxide indicator configured toundergo a color change suitable for visual assessment of whether thearticle has been exposed to the nitric oxide.

In some embodiments, a gNO indicator comprises one or more gNO-sensitivesubstances that can produce a detectable signal when exposed to acertain or any level of gNO in an environment. For example, thedetectable signal may be a color change of the gNO-sensitive substancewhich occurs upon exposure to gNO. Such a gNO indicator is suitable fora visual confirmation for sufficient exposure of the article to gNO.Exemplary gNO-sensitive substances include dyes such as, for example,4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM). It is to beunderstood that other gNO-indicators are contemplated in the context ofembodiments of the invention, such as other chemical indicators,electronic indicators, off-line indicators (a sample for measuring theexposure thereof to gNO at a site unrelated to the exposure site) andthe likes.

In some embodiments, a desiccant can be placed in the chamber to absorbhumidity, as illustrated in FIGS. 26-30 (desiccants 202, 302, 402). Thedesiccant can be disposed within the chamber and be configured to absorbhumidity from the ambient environment.

Suitable desiccants include, but are not limited to, Drierite®, silicagel, calcium sulfate, calcium chloride, montmorillonite clay, molecularsieves, etc. The desiccant can reduce the amount of humidity in theambient environment by about 75% to about 100%.

The charging device may also include at least one humidity indicator(see, indicator 208, 374 and 474 in FIGS. 32, 27 and 29 respectively)configured to indicate moisture saturation of the desiccant or humiditylevel in the chamber. A non-limiting example of a humidity indicator forindicating desiccant saturation include cobalt chloride (CoCl₂), whichundergoes a color change from blue (anhydrous state) to purple(CoCl₂.2H₂O) to pink (Co(H₂O)₆]Cl₂) as the absorption of waterincreases.

Devices which are configured for practicing any of processes asdescribed herein while generating reduced pressure in the chamberfurther comprise an outlet for generating a reduced pressure in thechamber.

In some embodiments, a package enclosing (or housing) the article isfurther included within the charging device. In some embodiments, thepackage is as non-gas permeable enclosure, utilized according toembodiments of the invention to provide a packaged gNO-sequesteringarticle as described herein.

FIG. 26 is a schematic illustration of an exemplary charging device 300,according to some embodiments of the present invention, which is fittedwith an inlet 345 for receiving gNO within chamber 337 to form gaseousnitric oxide-containing environment 306, valve 315 for closing outlet345, an outlet 326 for releasing ambient environment or gaseous nitricoxide-containing environment from chamber 337, valve 336 for closingoutlet 326, an article 301 disposed within chamber 337, and an optionalmoisture scavenger in the form of desiccant 302 disposed within chamber337 and configured to absorb an amount of humidity from the withinchamber 337. According to some embodiments of the present invention,inlet 345 can be used to connect to an external source of negativepressure, e.g. a vacuum pump (not shown in FIG. 26), for generating areduced pressure in chamber 337.

FIGS. 27-29 present schematic illustrations of exemplary chargingdevices similar with respect to the chamber, the environment, the inlet,the outlet, the valves and the article, to the charging device presentedin FIG. 26.

FIG. 27 is a schematic illustration of an exemplary charging device 300,as presented in FIG. 26, wherein chamber 337 also includes a surfacecoating 370 which protects the material of chamber 337 from chemicallyinteracting with gNO-containing environment 306, a gNO indicator 371configured to undergo a color change when article 301 has been exposedto gNO in environment 306, and humidity indicator 374 for signalingexposure of article 301 to humidity.

FIG. 28 is a schematic illustration of an exemplary charging device 400,according to some embodiments of the present invention, which is fittedwith an inlet 445 for receiving gNO within chamber 447 to form gaseousnitric oxide-containing environment 406, valve 415 for closing outlet445, an outlet 426 for releasing ambient environment or gaseous nitricoxide-containing environment from chamber 447, valve 436 for closingoutlet 426, and a non-gas permeable package 473 disposed within chamber447 and housing article 401. According to some embodiments of thepresent invention, inlet 445 or outlet 426 can be used to connect to anexternal source of negative pressure, e.g. a vacuum pump (not shown),for generating a reduced pressure in chamber 447.

FIG. 29 is a schematic illustration of an exemplary charging device 400,as presented in FIG. 28, wherein chamber 437 includes surface coating470 which protects the material of chamber 437 from chemicallyinteracting with gNO-containing environment 406, a gNO indicator 471configured to undergo a color change when article 401, havinggNO-sequestering surface 472 and housed within non-gas permeable package473 equipped with seal 490, has been exposed to gNO in environment 406,and humidity indicator 474 for signaling exposure of article 401 tohumidity.

While FIGS. 26-29 present exemplary charging devices fitted with only asingle inlet and a single outlet, the charging device may include aplurality of inlets and a plurality of outlets, as illustrated in FIG.30.

FIG. 30 presents a schematic illustration of an exemplary chargingdevice 500 having a chamber 137, inlet 125 with valve 11, inlet 135 withvalve 2, and inlet 145 with valve 15, inlets which may share a commontube 23 with valve 19 that connects to the chamber 137; outlet 126 withvalve 36 and connect to purge flow rotomoter 46, outlet 136 with valve41 and connect to purge flow rotomoter 45, outlets which may share acommon tube 33 that connects to the chamber 137. Separate inlets 125,135, 145 and outlets 126, 136 allow for gases (e.g. gNO, carrier gases)to enter and exit the chamber 137 at separate locations. Each of inlets125, 135, 145 may also allow a vacuum creating mechanism (not shown) tohave separate access to the chamber 137 for generating reduced pressuretherein.

Such a setup makes it convenient for an operator of the charging device137 to operate device 137 by removing the need to rehook vacuum, gNO andcarrier gas sources to a single inlet at different stages of theprocess. Using the configuration presented in FIG. 30 allows each inlet125, 135, 145 to be connected to a different source of gases or vacuum.For example, inlet 145 may serve as a vacuum port, inlet 125 may serveas an inlet for gNO or nitrogen gas, and inlet 135 may serve as apurging gas inlet for a carrier gas such as nitrogen gas, helium, argon,or any combination thereof.

The vacuum port 145 in FIG. 30 may be used to create a vacuum withinchamber 137 so that the gNO introduced to chamber 137 does not reactwith any gases or moisture already contained within chamber 137 orwithin the article disposed therein (not shown). Chamber 137 may bepressured to any suitable pressure, for example, once the article hasbeen disposed therein and chamber 137 has been sealed and evacuatedsubstantially from the ambient environment therein, chamber 137 may bepressurized to 50 psig of gNO or of gNO mixed with a carrier gas such ashelium, argon, nitrogen gas, and any combination thereof, helping tostabilize gNO.

Separate inlets 126, 136 in FIG. 30 may be connected by tubes to tworotometer kits 45, 46 which are flow meters that indicate flow rate andcan be operated in parallel.

The gaseous nitric oxide (gNO) sequestering articles presented hereincan be prepared (charged with gNO) and be ready for use well in advanceand kept in storage, or be prepared just prior to use, depending on theuse, conditions and other preferences. In some cases, the article can bekept for extended lengths of time in a sealed chamber which is designedfor charging its content with gNO, and then, prior to use, the deviceand article can be charged with gNO.

According to an aspect of embodiments of the invention, there isprovided a charging device which includes a sealed chamber having areduced pressure therewithin; and an article, as described herein (e.g.,a medical device), disposed within the chamber.

Such a chamber can be utilized, for example, for charging the articlewith gNO, as described herein, and thus can further comprise an inletconfigured for receiving a gaseous nitric oxide-containing environmentand an outlet for releasing said gaseous nitric oxide-containingenvironment.

In some embodiments, such a chamber further comprises an outlet used forgenerating the negative pressure within the chamber.

Such a chamber can further comprise a non-gas permeable enclosure inwhich the article is positioned, as described herein. The device canfurther comprise desiccants and NO-indicators as described herein.

It is expected that during the life of a patent maturing from thisapplication many relevant nitric oxide sequestering articles will bedeveloped and the scope of the phrase “nitric oxide sequesteringarticle” is intended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Materials and General Experimental Methods

The preparation of various articles, objects and devices, releasablysequestering gaseous nitric oxide, according to some embodiments of thepresent invention, is presented below. For clarity, it is noted that theterms “impregnated”, “charged”, “dosed” and “treated” are usedinterchangeably hereinbelow and throughout to denote an articlereleasably sequestering gaseous nitric oxide therein.

Nitric Oxide Impregnation—General Procedure I

Articles are exposed to nitric oxide (Airgas Specialty Gases, Chicago,Ill.) in nitrogen (N₂) or argon (Ar) as a carrier gas (at aconcentration ranging from 800 ppm to 20,000 ppm, unless otherwiseindicated) for a period of 1-600 minutes or overnight (10-24 hours) in achamber, such as a stainless steel chamber. Articles are placed in achamber and nitric oxide is introduced into the chamber at a flow rateof from 0.5 liter/minute to 5 liter/minute (unless otherwise indicated),depending on the parameters and intended use of the device to beimpregnated.

NO impregnation of articles within a gas-permeable package is generallyeffected by placing the packaged article in the chamber and exposing thepackaged article to NO, as described herein.

Preparation of gNO-impregnated packaged articles with a non-gaspermeable package is generally effected by placing the article within anencasement made from the non-gas permeable packaging material whilekeeping the encasement open, exposing the article and the encasement togNO as described herein and thereafter sealing the encasement containingthe device within.

Nitric Oxide Impregnation—General Procedure II

Articles are exposed to nitric oxide (Airgas Specialty Gases, Chicago,Ill.) in nitrogen (N₂) or argon (Ar) as a carrier gas for a period of1-600 minutes or overnight (10-24 hours) in a sealed chamber, such as astainless steel chamber. Articles are placed in the chamber and thechamber is sealed. Prior to filling the chamber with nitric oxide, areduced (negative) pressure (e.g., of −10 PSI; about −0.7 atmospheres)is maintained by a vacuum pump connected to the outlet of the chamberfor a period of e.g., 1-15 minutes. When the desired negative pressureis reached, the pump is stopped and the outlet valve closed to maintainthe negative pressure inside the chamber. Thereafter, the inlet valveconnected to a nitric oxide-containing cylinder is opened to allow thenitric oxide in the carrier gas to flow in and fill the chamber. The gasinflux is stopped when a pressure of zero PSI is reached. After theexposure time, the gas inside the chamber is purged with air for atleast 5 minutes through nitric oxide absorbing filters. Thereafter thechamber is opened and the exposed article is kept at room temperature.

In a typical procedure, an article is placed in a stainless steelchamber as described herein and the chamber is sealed. Prior to fillingthe chamber with nitric oxide, a reduced (negative) pressure of −10 PSI(about −0.7 atmospheres) is maintained by a vacuum pump connected to theoutlet of the chamber for a period of 1-15 minutes. When the negativepressure is reached, the pump is stopped and the outlet valve closed tomaintain the negative pressure inside the chamber. Thereafter, the inletvalve connected to a nitric oxide-containing cylinder is opened to allowthe nitric oxide (Airgas Specialty Gases, Chicago, Ill.) in nitrogen(N₂) or argon (Ar) as a carrier gas (0.05-10 percent nitric oxide) toflow and fill the chamber. The gas influx is stopped when a pressure ofzero PSI is reached. After the exposure time, the gas inside the chamberis purged with air for at least 5 minutes through nitric oxide absorbingfilters. Thereafter the chamber is opened and the exposed article iskept at room temperature.

NO-Level Determination by Determination of Nitrites and Nitrates:

Nitric oxide has a half-life of a few seconds; therefore stablemetabolites of nitric oxide (nitrates and nitrites) were measured usingthe Griess test [Green et al., Anal Biochem 126:131-138] to determinenitric oxide concentration in an aqueous medium.

Absorbance was measured at 543 nm and was translated to nitriteconcentration using a standard curve prepared using samples with knownnitrite concentrations. Nitrite production was converted into parts permillion of nitric oxide as follows: nitric oxideppm=(46×[NO₂])×0.65×10⁻³. Each μM (μmol/liter) was multiplied by themolecular mass of nitrite (46 grams/mol). This value was converted toppm of nitric oxide, taking into account the difference in molecularweight (MW) between nitrites and nitric oxide (1:0.65 ratio), and themultiplication factor between grams and milligrams (10³).

Bacterial and Fungal Culture Preparation:

The clinical strains Enterococcus faecalis, Staphylococcus aureus, E.coli, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia), orstrains from the American Type Culture Collection (ATCC)—E. faecalis#29212, Staphylococcus saprophyticus #15305, Staphylococcus epidermidis#35984, E. Coli #25922, P. aeruginosa #14210, Acinetobacter baumanii#BAA-747, Candida albicans #14053) were used for the described studies.

Bacteria were grown to 0.5 McFarland standard, and 1-ml aliquots ofthese preparations each containing approximately 2.5×10⁸ CFU/ml werestored in vials at −70° C. On the day of the experiments, the freshstock was removed from the freezer, thawed, and 2 ml of media was added(Luria Broth (LB) for E. coli, P. aeruginosa, A. baumanii; SabouraudDextrose Broth for C. albicans; Brain Heart Infusion (BHI) for E.faecalis, S. saprophyticus, S. epidermidis). Cultures were furtherdiluted with same media to 10³ cuff/ml in volumes appropriate to theexperimental conditions.

Biofilm Formation Assay:

Biofilm formation assays were performed as described in O'Toole et al.,1998, Mol. Microbiol., 30:295-304. Briefly, an object sample was cutlengthwise and placed in vial with 4 ml of 1 percent weight per volumecrystal violet for 15 minutes. The vial was washed, and the solution wasreplaced with 4 ml of 95 percents ethanol. The extracted color wasmeasured by absorbance at 595 nm.

Discoloration Measurements:

Color measurements were obtained using a LabScan® XE spectrophotometer(HunterLabs; LabScan® XE is a registered trademark of Hunter AssociatesLaboratory, Inc. 11495 Sunset Hills Road Reston Va. 22090), and analyzedwith the easyMatch QC software (version 3.72) following manufacturers'protocols and the method disclosed by Englberger et al., 2006. Food NutrBull 27(4):281-91. Briefly, three nitric-oxide dosed (“treated”), orcontrol (“untreated”) samples were put into a small petri dishlengthwise and side-by-side with no space between them, and measurementsof each plate were done from four different angles (2 vertical and 2horizontal), in triplicates. The color values obtained included: L*—thelightness of the color (0 yields black and 100 indicates diffuse white);a*—position between red/magenta and green (negative values indicategreen while positive values indicate magenta; on a numeric scale rangefrom −100 to +100); and b*—position between yellow and blue (negativevalues indicate blue and positive values indicate yellow; on a numericscale range from −100 to +100).

Tensile Strength Measurements:

Tensile strength measurements were conducted using a TA.XTPLUS textureanalyzer, (Texture Technologies Corp), and then analyzed with theExponent 32 software (version 3.0.4.0). Treated and untreated 3-cmsamples of the studies object were stretched at a constant distanceusing the texture analyzer, following manufacturer's protocols. Themaximum force that was used to stretch a specific piece was measured,using a “Miniature tensile grip” probe, and total area under the curveof force as a function of time was thereby determined.

Hardness and Springiness Measurements:

Hardness and Springiness measurements were obtained using a TA-XT2Texture analyzer (Texture Technologies Corp), and analyzed with theXTRAD software (version 3.7). Hardness and springiness (or elasticity)were determined following manufacturers' protocols, and the methodsprovided by Zhu J. H. et al., 2009, Effect of Guar gum on therheological, thermal and textural properties of soybean β-conglyciningel, Int J Food Sci and Technol., 44: 1314-1322 and by Bourne M. C.,Food Texture and Viscosity Second Edition: Concept and Measurement,2002, Academic Press, New York. Hardness is defined as the peak forceduring the first compression cycle of the sample. Springiness, alsoreferred to as elasticity, is an indicator of how much the samplestructure is broken down by an initial compression under a controlledforce. The sample is pressed between two plates for a set distance (e.g.8 mm from initial contact of the top plate with the sample) at aconstant rate, such as 2.5 cm per minute, for two compression cycles. Ineach compression cycle a curve with the applied force increasing frombaseline to a maximum value was generated where the maximum compressiondistance is reached, followed by a reversal and reduction of appliedforce to a baseline value, and was plotted as a graph of force versustime. Springiness (L1/L2) is defined as a ratio of the time (in seconds)recorded between the initial application of compression force to themaximum value for the second compression cycle (L2), divided by the time(in seconds) recorded between the initial application of compressionforce to the maximum value for the first cycle (L1). The value referredto as “area” is the area under the curve of force/time for first peak[Rincker et al., 2007, Evaluating an objective method to measure freshpork loin firmness, Meat Sci., 77:213-219].

Example 1 NO-Impregnated Silicone Catheters

Six-mm diameter Folysil® silicone Foley catheters, (catalog no. AA6118;Coloplast® Corp. Minneapolis, Minn., USA) (Folysil® and Coloplast® areregistered trademarks of Coloplast A/S, Humlebaek, Denmark) wereaseptically cut into 2-cm sections and inoculated by immersion in 2 mlof one of 10³ CFU/ml, 10⁵CFU/ml, or 10⁸ CFU/ml E. coli. inocula for 30minutes. Inoculated catheter sections were subsequently dosed with20,000 parts per million of nitric oxide (at a flow rate of 30cc/minutes) during 1, 5, 15, 30, 60 or 120 minutes, using the generalprocedure I described hereinabove. Control catheter sections were notimpregnated with nitric oxide, but were stored in sterile sealed vials.

The nitric oxide-treated and untreated catheter segments were thenimmersed separately in 2 ml media and then incubated for 8 hours at 37°C. Aliquots of the incubation media were plated and numbers of CFUcounted. Bacterial loads were calculated as CFU per ml.

FIG. 1 shows the results of dosing the medical device samples withnitric oxide for 1, 5, 15, 30, 60 or 120 minutes, following incubationin a bacterial suspension of 10³, 10⁵ or 10⁸ CFU/ml for 8 hours at 37°C. The CFU/ml remaining in the suspension are shown with respect to dosetime and starting culture CFU/ml.

Table 1 presents the colony formation following exposure of cathetersegments to nitric oxide at various durations, wherein TNTC denotes “toonumerous to count” or over 10⁶ CFU/ml.

TABLE 1 15 30 60 120 Control 1 minute 5 minutes minutes minutes minutesminutes 10³ 8 × 10⁵ 0 0 0 0 0 0 CFU/ml 10⁵ 1 × 10⁶ 6 × 10⁵ 40 0 0 0 0CFU/ml CFU/ml CFU/ml 10⁸ TNTC TNTC TNTC 0 0 0 0

As can be seen in Table 1 and FIG. 1, 15 minutes of dosing with 30cc/minutes of 20,000 ppm nitric oxide (2 percents nitric oxide innitrogen) was sufficient time to impregnate the catheter segment withsufficient nitric oxide to kill all bacteria up to a 10³ inoculationwith E. coli. At lower CFU, e.g., 10³ CFU/ml inoculation, one minute wassufficient time to dose the catheter segment with sufficient nitricoxide to kill all bacteria. With a bacterial culture of 10³CFU/ml, 1 to5 minutes were sufficient times to dose the catheter segment withsufficient nitric oxide to provide antimicrobial effects as measured byreductions in CFU. Sterilization was observed with 15 or more minutesdosing times. These data demonstrate that nitric oxide exhibited anantimicrobial effect at all three concentrations of bacterial stockstested, and sterilized the catheter segments after 1 minute (10³) and 15minutes (10⁵ and 10⁸) of treatment.

Example 2 NO-Impregnated Silicone Catheters

A commercially-available catheter, such as a six-mm diameter Folysil®silicone

Foley catheters, (catalog no. AA6118; Coloplast® Corp. Minneapolis,Minn., USA) is aseptically cut into 2-cm sections and inoculated byimmersion in 2 ml of one of 10³ CFU/ml, 10⁵ CFU/ml, or 10⁸ CFU/ml E.coli. inocula for 30 minutes. Inoculated catheter sections aresubsequently dosed with nitric oxide using the general procedure IIdescribed hereinabove. Control catheter sections are not impregnatedwith nitric oxide, but are stored in sterile sealed vials.

The nitric oxide-treated and untreated catheter segments are thenimmersed separately in 2 ml media and incubated for 8 hours at 37° C.Aliquots of the incubation media are plated and numbers of CFU counted.Bacterial loads are calculated as CFU per ml.

Catheters impregnated with NO using general procedure II are found toeradicate bacteria at all of the tested bacteria concentrations.Sterilization is thus achieved by NO-impregnation using generalprocedure II.

Example 3 Nitric Oxide Loading in NO-Impregnated Catheters

Six-mm diameter Folysil® silicone Foley catheters were aseptically cutinto 2-cm sections and exposed to nitric oxide for 1, 5, 10, 20, 40, 60,120, 180 or 240 minutes, using general procedure I as describedhereinabove. The samples were then immersed in doubly-distilled waterand after 1 hour were assayed for nitrates and nitrites, as describedhereinabove. All calculations were done per 1 cm of catheter.

FIG. 2 presents a plot of nitrite in μmol per cm of catheter relative todosing time.

Table 2 presents the raw data of release of nitrates and nitrites fromnitric oxide dosed catheter sample over varying dose times, wherein“Abs.” denotes absorbance, and “Abs average” denotes the averageabsorbance measured from duplicate sampling.

TABLE 2 NO2 conc. μmol NO2 NO Time Abs NO₂ in μmol/ ppm/ (min) Abs. Abs.average (μM) solution cm cm 1 0.066 0.069 0.07 25.96 0.08 0.04 0.39 50.275 0.274 0.27 105.58 0.32 0.16 1.58 10 0.398 0.364 0.38 146.54 0.440.22 2.19 20 0.491 0.53 0.51 196.35 0.59 0.29 2.94 40 0.531 0.574 0.55212.50 0.64 0.32 3.18 60 0.898 0.912 0.91 348.08 1.04 0.52 5.20 1201.265 1.312 1.29 495.58 1.49 0.74 7.40 180 1.689 1.557 1.62 624.23 1.870.94 9.33 240 2.096 1.618 1.86 714.23 2.14 1.07 10.68

As can be seen in Table 2 and FIG. 2, the amount of sequestered nitricoxide, reflected by the amount of nitrites, correlates to the time ofexposure, showing a tendency for saturation at exposure periods over 240minutes.

Example 4 Nitric Oxide Loading in NO-Impregnated Catheters

In order to compare nitric oxide charge using general procedures I andII as described hereinabove, 2-cm sections of commercially availablecatheters as described herein are placed in a chamber as describedherein and are exposed to 20,000 ppm nitric during 240 minutes,according to general procedure I described hereinabove.

In a separate assay, 2-cm sections of commercially available cathetersas described herein are placed in a chamber as described herein, thechamber is sealed and reduced pressure is generated in the chamber, andnitric oxide is then allowed to fill the chamber, according to generalprocedure II described hereinabove.

The samples of each assay are then immersed in doubly-distilled waterand after 1 hour are assayed for nitrates and nitrites, as describedhereinabove, using the Griess reagent, and nitrites and nitratesconcentrations are determined.

Calculations are done per 1 cm of catheter and show NO charge that ishigher by at least 20%, or at least 50%, compared to cathetersimpregnated with nitric oxide using general procedure I as describedhereinabove.

Example 5 Effects of Storage of NO-Impregnated Catheters onAntimicrobial Activity

To assess the effect of storage on antimicrobial activity after dosingarticles with nitric oxide, dosed sections of silicone catheters werestored in sealed containers containing air or water for one week.

Six-mm diameter Folysil® silicone Foley catheters were aseptically cutinto 2-cm sections and dosed with nitric oxide overnight (10-24 hours)as described in general procedure I described hereinabove.

After storage, the catheter sections were immersed in suspensions of 10³CFU/ml E. coli for 1 minute. After transfer to PBS and incubation for 3hours or 24 hours, no bacterial growth was observed in the aliquotsprepared from nitric oxide sequestering catheter sections. However,significant bacterial growth (10⁸ CFU/ml) occurred in the aliquotsprepared from the control catheter.

FIGS. 3A-B show images of representative plates of control and nitricoxide-eluting samples (FIG. 3A); and viable counts of CFU in triplicate(FIG. 3B), wherein the hatched bars represent data taken from controlexperiments, and the checkered bars represent data taken from the nitricoxide sequestering samples.

As can be seen in FIGS. 3A-B, after a week of storage in air or water,catheter section samples sequestering nitric oxide had absorbedsufficient nitric oxide to subsequently elute sufficient nitric oxide todemonstrate antimicrobial and sterilizing activity when exposed tobacterial cultures.

These results demonstrate the capacity of nitric oxide-dosed medicaldevices to elute nitric oxide levels sufficient for self-sterilizationafter being stored for 1 week in either air or water.

Example 6 Effects of Storage of NO-Impregnated Catheters onAntimicrobial Activity

Commercially available catheters, such as six-mm diameter Folysil®silicone Foley catheters are aseptically cut into 2-cm sections anddosed with nitric oxide as described in general procedure II describedhereinabove.

After a week of storage in air or water storage, the catheter sectionsare immersed in suspensions of 10³ CFU/ml E. coli for 1 minute. Aftertransfer to PBS and incubation for 3 hours or 24 hours, no bacterialgrowth is observed in the aliquots prepared from nitric oxidesequestering catheter sections.

Example 7 Effects of NO-Impregnated Catheters on Urinal Bacterial Flora

Six-mm diameter Folysil® silicone Foley catheters were dosed with 20,000ppm nitric oxide at a flow rate of 30 cm/minute for 24 hours, accordingto the general procedure I described hereinabove.

Using a dynamic urine flow model system, urine (2 liters) was collectedfrom male volunteers and placed in two 1 liter sterile plasticcontainers. Two ml of bacterial culture (10² CFU/ml) were inoculatedinto each container.

One container was circulated through nitric oxide-dosed catheter tubesand the control container was circulated using untreated catheter tubes.Urine samples from each of the containers were re-circulated in aseparate closed system using a flow rate of 1.5 ml/minute (Rabbit-Plusperistaltic pump; Rainin) through the catheters, for 24 hours at 37° C.Bacterial levels in the control and treated containers reached 10⁸ and10⁷ CFU/ml after 24 hours, respectively.

The catheters were then aseptically removed, washed with sterile water,cut into 3-cm pieces, and processed as follows:

A biofilm formation assay was performed as described hereinabove witheach catheter piece. FIG. 4A shows the absorbance at 595 nm of thecrystal violet that was attached to the catheter pieces after extractionwith ethanol. The hatched bars are data from the control, while thecheckered bars are data from the impregnated catheters. The error barsindicate standard deviations.

As can be seen in FIG. 4A, more than twice the amount of biofilm matrixwas formed on the luminal surface of the control catheter compared withthe impregnated catheter when using crystal violet.

In order to measure biofilm-embedded bacteria, each piece was cutlengthwise, washed and put into 4 ml sterile water.

Following sonication for 30 seconds, 1 μl and 10 μl of water surroundinga selected catheter piece were plated on LB plates and incubated at 37°C. for 24 hours.

FIG. 4B presents images demonstrating biofilm-embedded bacteria withinthe biofilms on control and impregnated catheters, grown in LB platesfrom the 1 μl (bottom) and 10 μl (top) of water surrounding a selectedcatheter piece.

As can be seen in FIG. 4B, after the removal of matrix-bound bacteria bysonication, there were no bacteria on the impregnated catheter surface,and there were an average of 1.210 CFU/ml on the control piece.

Example 8 Effects of NO-Impregnated Catheters on Urinal Bacterial Flora

Commercially available catheters, such as six-mm diameter Folysil®silicone Foley catheters are dosed with nitric oxide, according toeither general procedure I or general procedure II describedhereinabove.

Using a dynamic urine flow model system, urine (2 liters) is collectedfrom male volunteers and placed in two 1 liter sterile plasticcontainers. Two ml of bacterial culture (10² CFU/ml) are inoculated intoeach container.

One container is circulated through catheter tubes dosed by nitric oxideusing general procedure I, one container is circulated through catheterstubes dosed by nitric oxide using general procedure II, and the controlcontainer is circulated using untreated catheter tubes. Urine samplesfrom each of the containers are re-circulated in a separate closedsystem using a flow rate of 1.5 ml/minute (Rabbit-Plus peristaltic pump;Rainin) through the catheters, for 24 hours at 37° C. Bacterial levelsin the control and treated containers are then measured.

The catheters are then aseptically removed, washed with sterile water,cut into 3-cm pieces, and processed as follows:

A biofilm formation assay is performed as described hereinabove witheach catheter piece.

Absorbance measurement show that NO-treated catheters exhibit asubstantially lower amount of biofilm matrix formed on the luminalsurface thereof compared with control catheters. Catheters impregnatedwith NO using general procedure II exhibited an amount of biofilm matrixformed on the luminal surface which is lower by 20-50% compared tocatheters impregnated with NO using general procedure I.

In order to measure biofilm-embedded bacteria, each piece is cutlengthwise, washed and put into 4 ml sterile water. Following sonicationfor 30 seconds, 1 μl and 10 μl of water surrounding a selected catheterpiece are plated on LB plates and incubated at 37° C. for 24 hours.

After the removal of matrix-bound bacteria by sonication, there are nobacteria on the NO-impregnated catheter surface.

Example 9 Preparation and Characterization of unpackaged NO-ImpregnatedCatheters

Six-mm diameter Folysil® silicone Foley catheters were cut into 3 cmsections, referred to as “samples” or “medical device samples”, and weredosed with nitric oxide for 24 hours at a flow rate of 50 ml/minute asdescribed in general procedure I hereinabove. The physical properties ofthe samples were tested immediately upon NO dosing and after one-weekstorage either in water or in air (in a sealed plastic vial at roomtemperature (about 20° C.).

Measurements after Dosing:

Color measurements were conducted as described above, using three 3-cmnitric-oxide treated catheter sections and three untreated cathetersections as control.

Table 3 presents the results of the discoloration assessment ofNO-treated samples after 24-hour nitric oxide dosing, and of untreatedcontrol samples.

For all the following tables, T-test of (−) denotes ‘no statisticaldifference when P<0.05’, and (+) denotes ‘statistical difference whenP<0.05’.

TABLE 3 NO- T-test P Parameter untreated treated (P < 0.05) valuevertical average L* 25.9 ± 1.4  25.5 ± 0.46 − 0.56 average a* 1.29 ±0.42 1.06 ± 0.17 − 0.24 average b* 0.69 ± 0.13 0.71 ± 0.18 − 0.89horizontal average L* 25.7 ± 1.02 26.1 ± 0.47 − 0.52 average a* 1.36 ±0.4  1.09 ± 0.19 − 0.15 average b* 0.86 ± 0.27 1.50 ± 0.16 + 0.006

As can be seen in Table 3, no significant change in color was observedduring storage; however, the NO-treated samples were indistinguishablefrom the untreated samples in a visual inspection thereof.

Table 4 presents the results obtained in the tensile strengthmeasurements of NO-treated and untreated samples.

TABLE 4 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce (g) 3.17 ± 0.1  3.13 ± 0.06 − 0.38 Area 14.79 ± 0.79 14.61 ± 0.42− 0.64

Table 5 presents the results obtained in the hardness and springinessmeasurements of NO-treated and untreated samples.

TABLE 5 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce-1 (g) 23.8 ± 0.43 23.8 ± 0.42 − 0.95 Max force-2 (g) 21.1 ± 0.3421.2 ± 0.39 − 0.53 Area 32.0 ± 0.78 32.2 ± 0.38 − 0.5 L2/L1* 0.94 ± 0.020.95 ± 0.01 − 0.673

As can be seen in Table 4 and Table 5, no significant differences wereobserved in tensile strength, hardness or springiness of the NO-treatedmedical device samples compared to the untreated control samples,following 24 hour of dosing with nitric oxide as described.

Measurements after Dosing and Wet Storage:

Table 6 presents the results of the discoloration assessment ofNO-treated samples after 24-hour nitric oxide dosing and one week ofstorage in water, and of untreated control samples after one week ofstorage in water.

TABLE 6 NO- T-test P Parameters untreated treated (P < 0.05) valuevertical average L* 26.7 ± 0.8 29.6 ± 0.8 + <0.0001 average a*  1.5 ±0.3 0.85 ± 0.3 + <0.0001 horizontal average L* 25.6 ± 0.8 28.5 ± 0.8 +0.0001 average a* 1.57 ± 0.2 0.84 ± 0.3 + 0.0006

The “b*” values exhibited high variability for both sets of samples,showing statistically different results (data not shown).

As can be seen while comparing Table 3 and Table 6, a slight change inlightness (Value “L*”) was observed in the NO-treated samples after oneweek of storage in water.

Table 7 and Table 8 present the results of the tensile strength andhardness-springiness measurements, respectively, as obtained forNO-treated and untreated samples after storage in water for one week.

TABLE 7 NO- T-test P Parameter untreated treated (P < 0.05) value Force(g)  3.1 ± 0.1  3.3 ± 0.05 + 0.001 Area 14.8 ± 0.8 15.9 ± 0.6 − 0.05

TABLE 8 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce-1 (g) 23.5 ± 0.8 24.1 ± 0.7 − 0.26 Max force-2 (g) 20.8 ± 0.8 21.2± 0.5 − 0.3 Area 30.4 ± 0.2 30.6 ± 0.3 − 0.56 L2/L1 0.931 ± 0.01 0.926 ±0.02 − 0.39

As can be seen in Tables 6-8, following 1-week storage in water, nosignificant differences were observed in tensile strength, hardness andspringiness, relative to controls.

Measurements after Dosing and Dry Storage:

Table 9 presents the results of the discoloration assessment ofNO-treated samples after 24-hour nitric oxide dosing and one week ofstorage in air, and of untreated control samples after one week ofstorage in air.

TABLE 9 NO- T-test P Parameter untreated treated (P < 0.05) valuevertical average L 25.7 ± 1.5 25.7 ± 2.2 − 0.97 average a  1.3 ± 0.3 1.1 ± 0.2 − 0.09 horizontal average L 25.2 ± 1.5 25.7 ± 2   − 0.67average a 1.25 ± 0.2 0.91 ± 0.1 + 0.006

As can be seen while comparing data in Tables 3 and 9, a slight changein “a*” value was observed in the NO-treated samples after one weekstorage in air.

Table 10 and Table 11 present the results of the tensile strength andhardness-springiness measurements, respectively, as obtained forNO-treated and untreated samples after storage in air for one week.

TABLE 10 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce (g)  3.3 ± 0.07  3.4 ± 0.1 − 0.06 Area 16.3 ± 0.7 17.3 ± 0.8 −0.06

TABLE 11 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce-1 (g) 24.0 ± 0.8 25.7 ± 0.5 + 0.003 Max force-2 (g)  21.2 ± 0.0223.1 ± 0.4 + 0.003 Area 32.9 ± 1.2 34.7 ± 0.8 + 0.03 L2/L1 0.935 ± 0.010.935 ± 0.02 − 0.95

As can be seen in Tables 10 and 11, following 1-week storage in air, nosignificant differences were observed in tensile strength andspringiness, relative to controls. A change in the hardness (forcevalues) was observed.

Example 10 Preparation and characterization of Packaged NO-ImpregnatedCatheters

Individually packaged (in a gas-permeable package) Folysil® catheters(Coloplast® AA6118) were treated with nitric oxide (20,000 ppm) for 24hours, at a flow rate of 1 liter/minute using general procedure Idescribed hereinabove. After NO-treating, the packaged catheters werestored for one month at ambient temperature and humidity. Followingstorage, catheters were analyzed for color, tensile strength andhardness-springiness as described hereinabove.

The results are presented in Tables 12-14, whereas Table 12 presentscolor measurements, Table 13 presents tensile strength, and Table 14presents results of the hardness-springiness measurements.

TABLE 12 NO- T-test P Parameter untreated treated (P < 0.05) valuevertical average L 25.4 ± 1.1  25.8 ± 0.92 − 0.66 average a 1.35 ± 0.421.21 ± 0.17 − 0.29 average b 0.69 ± 0.13 0.71 ± 0.18 − 0.81 horizontalaverage L 25.4 ± 0.46 25.6 ± 1.10 − 0.62 average a 1.31 ± 0.4  1.28 ±0.19 − 0.48 average b 0.72 ± 0.27 1.14 ± 0.16 + 0.009

TABLE 13 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce(g) 3.21 ± 0.1  3.15 ± 0.06 − 0.36 Area 14.79 ± 0.79 14.61 ± 0.42 −0.64

TABLE 14 NO- T-test P Parameter untreated treated (P < 0.05) value Maxforce-1 (g) 23.7 ± 0.43 23.6 ± 0.42 − 0.42 Max force-2 (g) 21.1 ± 0.3821.2 ± 0.34 − 0.64 Area 31.9 ± 0.71 32.0 ± 0.28 − 0.77 L2/L1 0.95 ± 0.010.95 ± 0.01 − 0.95

As can be seen in Tables 12-14, following one month of storage in apermeable package, no significant changes in color, hardness,springiness and tensile strength were observed between the NO-treated,packaged and stored catheters relative to untreated, packaged and storedcatheters.

Example 11 Preparation and Characterization of Additional PackagedNO-Impregnated Catheters

Individually packaged (with gas-permeable package) catheters, denoted A,B, C as described below, were treated with nitric oxide (20,000 ppm) for24 hours, at a flow rate of 1 liter/minute as described in generalprocedure I hereinabove. After NO-treating, the packaged catheters werestored for one month at ambient temperature and humidity. Followingstorage, catheters were analyzed for color, tensile strength andhardness-springiness as described hereinabove.

Exemplary catheter A is a silicone coated, latex based device: SILASTIC®by Dow Corning Corp., Midland, Mich. USA, Foley catheter 16 Fr. Bard® byC. R. Bard Inc., Murray Hill, N.J., USA.

Exemplary catheter B is a silicone coated, latex based device: bardia,Foley catheter, 2-way silicone elastomer coated, 20 Ch/Fr (6.7 mm) byBard®.

Exemplary catheter C is an all-silicone device: Catheter urethraldrainage Foley 2 way 30 CC balloon all silicone, 18 Fr. Bard®.

The results are presented in Tables 15-17, whereas Table 15 presentscolor measurements, Table 16 presents tensile strength, and Table 17presents results of the hardness-springiness measurements.

TABLE 15 NO- T-test Sample Parameter untreated treated (P < 0.05) Pvalue A vertical average L   60 ± 0.7   58 ± 0.8 − 0.06 average a −27.9± 0.3 −26.4 ± 0.3 + <0.0001 average b  12.8 ± 0.4  18.2 ± 0.5 + <0.0001horizontal average L   59 ± 0.5   58 ± 0.7 − 0.12 average a −27.6 ± 0.4−26.3 ± 0.2  + <0.0001 average b 11.8 ± 1   17.1 ± 0.8 + <0.0001 Bvertical average L  35 ± 1  36 ± 1.5 − 0.132 average a  12.7 ± 0.2  15.8± 0.7 + <0.0001 average b   22 ± 0.8  29 ± 2.2 + <0.0001 horizontalaverage L   38 ± 0.6  36 ± 1.5 − 0.43 average a  12.6 ± 0.4  15.8 ±0.7 + <0.0001 average b   22 ± 0.6  29 ± 2.4 + <0.0001 C verticalaverage L   38 ± 0.9  38 ± 1.4 − 0.98 average a  −0.9 ± 0.16 −1.4 ±0.2 + 0.003 average b  −5.8 ± 0.6 −4.6 ± 0.7 + <0.0001 horizontalaverage L   38 ± 1.2  37 ± 1.3 − 0.83 average a  −0.8 ± 0.2 −1.3 ± 0.3 +0.003 average b   −6 ± 0.5 −4.9 ± 0.9 + <0.0001

As can be seen in Table 15, lightness (“L” value) did not change afterimpregnation with nitric oxide in all three catheter sample types, while“a” and “b” values did demonstrate a significant color change.

TABLE 16 Sam- NO- T-test P Parameter ple untreated treated (P < 0.05)value Max force (g) A  1.1 ± 0.06   1 ± 0.04 − 0.164 B 1.56 ± 0.07 1.52± 0.06 − 0.06 C 2.8 ± 0.2  3.5 ± 0.07 + <0.0001 Area A 5.4 ± 0.3  5 ±0.3 − 0.07 B 7.9 ± 0.4 7.2 ± 0.3 + 0.001 C 14.2 ± 1.6  18.3 ± 0.4  +<0.0001

As can be seen in Table 16, tensile strength of the all-siliconecatheter C demonstrated a significantly greater maximum force comparedto the other catheters.

TABLE 17 Sam- NO- T-test P Parameter ple untreated treated (P < 0.05)value Max A  7.3 ± 0.3  6.1 ± 0.1 + <0.001 force-1 (g) B 11.1 ± 0.8 10.6± 0.5 − 0.32 C  13 ± 0.8 14.8 ± 0.7 + 0.003 Max A   7 ± 0.3  5.8 ± 0.1 +<0.001 force-2 (g) B 10.4 ± 0.8 10.1 ± 0.8 − 0.44 C  11 ± 0.8 12.2 ±0.8 + 0.024 Area A 13.6 ± 0.4  9.9 ± 0.4 + <0.001 B 17.2 ± 1.2 16.7 ±0.9 − 0.37 C 18.4 ± 0.8 20.9 ± 0.8 + 0.0003 L2/L1 A  0.84 ± 0.001 0.79 ±0  + <0.001 B  0.93 ± 0.008  0.94 ± 0.01 − 0.58 C  0.94 ± 0.005  0.94 ±0.002 − 0.91

As can be seen in Table 17, springiness did not vary significantly forthe catheters tested; however, following nitric oxide treatment,catheters A and C demonstrated a change in hardness.

For the parameters measured, while some changes are indicated to bestatistically significant (e.g., there is a difference in the color,hardness, springiness or tensile strength between the treated andcontrol samples), this may be small, and not indicative of an alterationin physical properties of the medical device that would otherwise renderit unsuitable for the intended application after dosing with nitricoxide.

It is noted herein that a visible color change due to nitric oxidedosing may be useful as an indicator of treatment, and/or sterility ofthe medical device, as further explained in detail hereinabove.

Example 12 Preparation and Characterization of Packaged and UnpackagedNO-Impregnated Catheters

Commercially available catheters such as six-mm diameter Folysil®silicone Foley catheters and the catheters described in Example 11hereinabove, are cut into 3 cm sections, and are dosed with nitric oxideeither using general procedure I described hereinabove or using generalprocedure II as described hereinabove. The physical properties of thesamples are tested immediately upon NO dosing and after one-week storageeither in water or in air (in a sealed plastic vial at room temperature(about 20° C.).

Color measurements and measurements of tensile strength, andhardness-springiness are performed after dosing and upon one weekstorage in air or water.

In a different set of experiments, individually packaged commerciallyavailable catheters such as Folysil® catheters (Coloplast® AA6118) andthe catheters described in Example 11 hereinabove are treated withnitric oxide (20,000 ppm) for 24 hours, at a flow rate of 1 liter/minuteusing general procedure I described hereinabove, or according to generalprocedure II described hereinabove.

After NO-treating, the packaged catheters are stored for one month atambient temperature and humidity. Following storage, catheters areanalyzed for color, tensile strength and hardness-springiness asdescribed hereinabove.

Example 13 Biofilm Sterilization by Gaseous NO

A biofilm was established in each well of a 6-well plate and the time oftreatment with nitric oxide gas for eradication of the biofilm wastested using the biofilm formation assay described hereinabove.

Briefly, 3 ml of LB media were placed in each well of 12 6-well platesand then each well was inoculated with 10 μl of stock bacteria (E. Colior A. baumanii (6 plates for each bacterial type). Plates were incubatedwith agitation for 5 days at 37° C. to establish a biofilm in each well.The media was removed and each well washed twice with sterile saline.

Plates with established biofilms (one for each bacterial type) weretreated with nitric oxide (20,000 ppm, 1 liter/minute flow rate) for 5,10, 30, 60 or 120 minutes. Control plates were kept at room temperature,without treatment.

Three of the six wells for each plate were stained with crystal violetto confirm biofilm formation. The biofilm in the remaining 3 wells ofeach plate was re-suspended (with 30 seconds sonication) in 5 ml sterilesaline. Aliquots of 1, 10 and 100 μl of each sample were plated incompartmentalized (3-section) petri plates.

FIG. 5A shows the results of the 1, 10 and 100 μl aliquots for E. Coli,while

FIG. 5B shows the results of the 1, 10 and 100 μl aliquots for A.baumanii.

As can be seen in FIGS. 5A-B, a biofilm was established for all wells(E. coli and A. baumanii). Absorbance (595 nm) of the crystalviolet-stained wells was found to be 0.7 to 1 with no significantdifference observed between control and nitric oxide treated wells.

Control plates demonstrated bacterial growth proportional to theinoculum, while for all nitric-oxide treated plates (for eitherbacterial species), no colonies were observed.

Example 14 Antimicrobial Activity of NO-Impregnated Catheters

NO-dosed catheters were prepared as described in general procedure Ihereinabove. Untreated catheters were used as control.

One and a half (1.5) ml of 10³ CFU/ml inoculum of each of the testedstrains of bacteria or fungi (ATCC strains or clinical isolates) wereincubated with 2 cm of catheter section at 37° C. for 24 hours. At timepoints of 0 and 24 hours, tubes were vortexed and aliquots were platedon LB agar then incubated at 37° C. for 24 hours. Colony-forming unitswere counted and final bacterial load calculated as CFU/ml.

The following bacterial strains obtained from the ATCC were tested:

Enterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus#15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984),Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210(P.a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA-747), andCandida albicans (C.a. #14053).

The following bacterial clinical isolates were tested: Enterococcusfaecalis (E.f.), Staphylococcus aureus (S.a.), E. coli (E.c.), P.aeruginosa (P.a.), Stenotrophomonas maltophilia (S.m.)

FIGS. 6 and 7 present the data obtained for selected bacterial strainsobtained from the ATCC (FIG. 6) and for selected bacterial clinicalisolates (FIG. 7) in nitric oxide-dosed vs. control catheter sections.

As can be seen in FIGS. 6-7, all tested strains demonstrated a decreasein CFU when incubated with nitric oxide-treated catheter segments. Somespecies of bacteria or fungi (S. epidermidis #35984, E. coli #25922, P.aeruginosa #14210, C. albicans #14053) were eradicated followingincubation with the nitric oxide-treated catheter segments, whereasothers (clinical isolates of E. faecalis, S. aureus, E. coli, P.aeruginosa, S. maltophilia; and E. faecalis #29212, S. saphrophyticus#15305, A baumanii #BAA-747) demonstrated a significant reduction in CFUremaining.

Example 15 Antimicrobial Activity of NO-Impregnated Catheters

NO-dosed catheters are prepared as described in general procedure I orII hereinabove. Untreated catheters are used as control.

One and a half (1.5) ml of 10³ CFU/ml inoculum of a bacterial of fungalstrain are incubated with 2 cm of catheter section at 37° C. for 24hours. At time points of 0 and 24 hours, tubes are vortexed and aliquotsare plated on LB agar then incubated at 37° C. for 24 hours.Colony-forming units are counted and final bacterial load calculated asCFU/ml.

All tested strains demonstrate a decrease in CFU when incubated withnitric oxide-treated catheter segments, with catheters impregnated withNO using general procedure II demonstrating an enhanced decrease in CFUcompared to catheters impregnated with NO using general procedure I.

Example 16 Bacterial Colonization on Surfaces of NO-ImpregnatedCatheters

Catheter sections were immersed for 24 hours in 10³ CFU/ml of each ofthe ATCC and clinical strains of bacteria or fungi described in Example15 hereinabove. Catheter sections were washed twice with steriledistilled water and then aseptically transferred to agar platescontaining the appropriate growth media for the particular strain. Eachsection was rolled once on the plate and the plate incubated at 37° C.overnight.

Table 18 presents the data inspected for the presence of bacteria on thesurface of nitric oxide-treated catheter compared to untreated controlsamples, wherein sections denoted “2” exhibited the same growth densityas that of the control; “1” exhibited less growth density compared tocontrol; and “0” exhibited no growth.

TABLE 18 Untreated catheter NO-treated catheter section (control)section E. faecalis #29212 2 2 Staphylococcus saprophyticus 2 1 #15305Staphylococcus epidermidis 2 1 #35984 E. coli #25922 2 0 P. aeruginosa#14210 2 0 A. baumanii #BAA-747 2 0 C. albicans #14053 2 0 E. faecalis 21 S. aureus 2 1 E. coli 2 0 P. aeruginosa 2 0 S. maltophilia 2 0

As can be seen in Tables 18, reduction or complete eradication ofcolonizing bacteria on the surface of the NO-treated catheter wasdemonstrated for the majority of the tested strains tested.

Example 17 Bacterial Colonization on Surfaces of NO-ImpregnatedCatheters

NO-dosed catheters are prepared as described in general procedure I orII hereinabove. Untreated catheters are used as control.

Catheter sections are immersed for 24 hours in 10³ CFU/ml of a bacterialor fungal strain, washed twice with sterile distilled water and thenaseptically transferred to agar plates containing the appropriate growthmedia for the particular strain. Each section is rolled once on theplate and the plate incubated at 37° C. overnight.

Reduction or complete eradication of colonizing bacteria on the surfaceof the NO-treated catheter is generally demonstrated, with enhancedreduction of colonizing bacteria being inspected in cathetersimpregnated with NO using general procedure II described hereinabove.

Example 18 Biofilm Formation in Urine on Surfaces of NO-ImpregnatedCatheters

Measurements of Biofilm Formation and Biofilm-Embedded Bacteria inNO-Treated Catheters:

To simulate a static, clinically-relevant milieu, such as found forexample in an installed urinary catheter, catheters were incubated inurine instead of media. Urine was collected from male volunteer andplaced into sterile vial (1.8 ml in each vial). 200 μl of 10³ ofbacteria were inoculated into each vial to reach a final concentrationof 10² CFU/ml.

The following bacteria were tested: E. faecalis #29212, S. saprophyticus#15305, S. epidermidis #35984, E. coli #25922, P. aeruginosa #14210, A.baumanii #BAA-747, C. albicans #14053,

NO-treated and untreated control catheters (2 cm length) were placedinto each of the vials. Vials were incubated for 72 hours at 37° C.Thereafter catheters were aseptically removed, washed with sterilewater, cut into 1 cm pieces, and the formation of a biofilm was assessedas described hereinabove.

FIG. 8 presents the relative biofilm formation on luminal surfaces ofnitric oxide-sequestering catheter sections following 72 hour incubationin urine inoculated with 10³ CFU/ml of the tested bacteria.

As can be seen in FIG. 8, biofilm formation was reduced for all testedstrains where catheters were treated with nitric oxide before incubationin urine.

In order to measure biofilm-embedded bacteria, each lengthwise-cut pieceof catheter was washed and put into 4 ml sterile water. Followingsonication for 30 seconds, samples were plated on compatible agar platesin triplicates and incubated at 37° C. for 24 hours.

FIG. 9 presented the data obtained for the growth of biofilm-embeddedbacteria on nitric oxide-sequestering catheter sections following 72hours incubation in urine inoculated with 10³ CFU/ml bacteria.

As can be seen in FIG. 9, bacteria embedded within the biofilm wereeradicated in 6 of the 7 species tested, with only P. aeruginosaexhibiting colony growth.

Scanning Electron Microscopy assessment of Biofilm:

Catheter sections of 1 cm length from the above-described biofilmstudies, inoculated with Staphylococcus epidermidis or A. baumanii, wereused. Samples were washed 3 times with sodium cacodylate (0.1 M, pH 7.4)then post-fixed in 0.5% tannic acid and 1% buffered OsO₄ using a Pelco3450 laboratory microwave (Redding, Calif., USA). The samples wererinsed 3 times with water, dehydrated in a graded ethanol series, anddried in a Tousimis 815B critical point drier (Tousimis, Rockville, Md.,USA) or chemically dried with hexamethyldisilazane (Sigma, St. Louis,Mo., USA) mounted on aluminum SEM stubs and coated with 8 nm of goldusing a Cressington 208HR sputter coater. Catheter pieces were imagedusing a Hitachi 54700 FESEM (Hitachi, Tokyo, Japan).

FIGS. 10A-C present scanning electron micrographs Staphylococcusepidermidis biofilms on NO-sequestering and untreated control cathetersections, wherein untreated control sample at magnification 2.5 k (insetat 20.0 k) is shown in FIG. 10A, untreated control sample atmagnification 2.5 k (right inset at 15 k, left inset at 20 k) is shownin FIG. 10B and nitric oxide treated catheter sample at magnification1.5 k is shown in FIG. 10C.

FIGS. 11A-C present scanning electron micrographs of A. baumanii (ATCC#BAA-747) biofilms on nitric oxide-sequestering and untreated controlcatheter sections, wherein untreated control sample at magnification 2.5k (inset at 15 k) is shown in FIG. 11A, untreated control sample atmagnification 1 k (inset at 5 k) is shown in FIG. 11B and nitric oxidetreated catheter sample at magnification 1 k is shown in FIG. 11C.

As can be seen in FIGS. 10 and 11, little to no biofilm was observed onthe nitric oxide-treated catheter sections, while biofilm and embeddedbacteria were observe on the control untreated catheter sections.

Example 19 Comparison of Antiseptic Activity of NO-Impregnated UrinaryCatheters and Commercially Available Catheters

Antiseptic urinary catheters have recently become commercially availableand others are in the development stage. The efficacy of bothcommercially available and emerging urinary catheter technologies inrelation to their effects on bacteriuria caused by E. coli in vitro hasbeen studied.

Materials and Methods:

An untreated control and three different treated catheters were used inthis study, as follows:

Control: 6 mm diameter Folysil silicon Foley catheter Ch/Fr 18 (catalogno. AA6118; Coloplast Corp. Minneapolis, Minn.);

NOX: the same catheter as control after impregnation with NO asdescribed in general procedure I hereinabove;

AG: BARDEX I.C. silver-coated Anti-Infective Foley Catheter Ch/Fr 18(Catalog No. 0165SI18; Bard, Inc. Covington, Ga.); and

NFC: Release NF—Nitrofurazonecoated silicone Foley catheter Ch/Fr 16(Catalog No. 95216; Rochester Medical, Stewartville, Minn.).

Catheter pieces not treated with NO, used as controls, were stored in asterile sealed vial until use.

E. coli bacterial culture was obtained from American Type CultureCollection (ATCC #25922).

Bacteria were grown to 0.5 McFarland standard. 1 ml aliquots of grownbacteria containing approximately 2.5×10⁸ CFU/ml were stored at −70° C.On the day of the experiments the fresh stock was removed from thefreezer, thawed, and 2 ml of Luria Broth (LB) was added. Cultures werefurther diluted with LB to 10³ CFU/ml.

E. coli culture (2 ml) at a concentration of 10³ CFU/ml was added to avial containing a 2 cm piece of catheter and incubated for 24 hours at37° C. After 24 hours, samples were vortexed and plated (using a 10³time dilution) on LB agar plates and were then incubated at 37° C. for24 hours. The CFU was counted and calculated to represent the CFU perml.

E. coli culture (200 μl) at a concentration of 10³ CFU/ml was added to1.8 ml of urine (reaching a final concentration of 10² CFU/ml)containing a 2 cm section of a tested catheter and incubated at 37° C.for 72 hours. Urine was collected in a sterile vial from a malevolunteer on the day of the experiment. After 72 hours, samples werevortexed and plated onto LB agar plates and incubated at 37° C.overnight. The CFU were counted and calculated to represent the CFU/ml.

To qualitatively evaluate the colonization potential, 1, 10, and 100 μlof each sample were plated of a three compartment LB agar petri plate.Plates were incubated overnight at 37° C.

Catheter sections (2 cm) were immersed in tubes containing 3 ml of E.coli culture at 10³ CFU/ml and incubated at 37° C. for 24 hours. After24 hours, catheter sections were washed twice using 3 ml of sterilesaline (0.9% wt/v NaCl) and transferred aseptically to a LB agar plate.Each catheter section was rolled once on the plate then incubated at 37°C. for 24 hours.

Catheter pieces (2 cm each) were added, aseptically, to 1.8 ml of urine(collected as stated above) plus 200 μl of E. coli culture at 10³ CFU/mland incubated at 37° C. for 72 hours. After 72 hours, catheter pieceswere washed twice in 3 ml sterile saline then cut into two pieces ofequal size, aseptically. One half of each catheter section was added to1.5 ml of Crystal Violet dye in water (1% wt/v) for 15 minutes thenwashed twice in 4 ml distilled water (dH2O). Washed catheter pieces werethen added to 2 ml of 95% ethanol to relinquish crystal violet bound tothe surface of the catheter.

The absorbance at 595 nm of each ethanol sample was measured using aspectrophotometer and used as an indicator of biofilm formation.

The other half of each catheter section was added to 2 ml of steriledistilled water (dH₂O) and sonicated at a level of 5 for 30 seconds. Theextent of colonial E. coli released from each catheter was determined byplating each sample on LB agar plates and incubating at 37° C.overnight. The CFU were counted and calculated to represent the CFU perml.

Results:

FIG. 12 presents the comparative data obtained for E. coli growth inmedia containing pieces of the NOX, AG and NFC catheters versus mediafrom control catheter, after immersion of the catheters for 24 hours insuspension comprising 10³ CFU/ml and incubated for 24 hours at 37° C.

As can be seen in FIG. 12, after being immersed in bacterial culture for24 hours, the antibacterial activity of the NOX and NFC catheters vastlyexceeded that of both the untreated (control) and AG catheters. Controland AG catheters reached concentrations of 1.8×10⁸ and 2.1×10⁸ CFU/ml,respectively. NOX catheter pieces contained an average bacterialconcentration of 2.5×10² CFU/ml following the 24 hours incubation,revealing an effective reduction in the concentration of planktonic E.coli. No bacteria were observed in the sample containing NFC catheters.

FIG. 13 presents the comparative data obtained for E. coli growth inurine after 72 hours exposure to pieces of NOX, AG and NFC and controlcatheters. Within each three compartment LB agar petri plate 1, 10, and100 μl of each sample were plated and incubated overnight at 37° C.

As can be seen in FIG. 13, after being immersed in urine plus bacteriafor 72 hours, the solution containing the AG catheter had a similarbacterial concentration to the control, 2.0×10⁸ and 5.0×10⁷respectively, while the solutions containing the NOX and NFC catheterscompletely eradicated bacteria in urine.

The rolling of catheter pieces on LB agar plates provides a qualitativemeasure of the presence of bacterial colonization on the surface of eachcatheter.

FIG. 14 presents the comparative data obtained for E. coli colonizationon NOX, AG and NFC catheters versus control after immersion of cathetersfor 24 hours in suspension containing 10³ CFU/ml of E. coli. In each LBPetri dish a catheter was rolled over the surface and then incubated at37° C. overnight.

As shown in FIG. 14, both the control and AG catheters containedextensive colonization on the surface of the catheter, whereas forcatheters NOX and NFC, no bacterial colonization was observed.

Crystal Violet studies indicate the extent of biofilm formation on eachcatheter type. Each experiment was repeated three times with threereplicates in each one of the experiments. FIGS. 15A and 15B presentcomparative data of colonized biofilm formation on NOX, AG and NFCcatheters versus control after 72 hours of incubation, demonstrated byabsorbance at 595 nm of the Crystal Violet that was attached to thecatheter pieces after extraction of color with ethanol (FIG. 15A) andthe bacterial growth from the biofilms from the different catheters(FIG. 15B).

As can be seen in FIG. 15A, the AG catheter showed the highest averageabsorbance indicating extensive biofilm formation. The AG catheterrendered an average absorbance 8-10 times greater than all othercatheter types, even the control. The NOX catheter had half theabsorption of the control and the NFC catheter had 40% less. Thisindicates that both the NOX and NF catheters had less biofilm formationon their surface when compared to the control. Comparative ANOVA test(p<0.005) shows that all three tested catheters were significantlydifferent from the control and NF and NOX were found to be significantlydifferent from AG. FIG. 15B shows the amount of colonized bacteriareleased from the catheter surface. The NOX and NFC catheters provedeffective at eradicating bacteria embedded within the biofilm whereasthe control and AG catheters produced bacteria concentration of 7.5×10⁴and 9.8×10² CFU/ml, respectively.

The obtained data demonstrate that NF and NO impregnated urinarycatheters possess similar antimicrobial properties, whereby the silvercoated catheter was found to be effective.

NO impregnated catheters are shown to be comparable to antimicrobialcoated urinary catheters in their level of antimicrobial activity. NOcoated catheters prevented E. coli growth in urine for 72 hours, same asthe NF catheters.

Antibiotics and NO kill bacteria in different ways, thus, theirspecificity is not the same. Antibiotics are specific to an organism ora group of organisms whereas NO is not. NF was found to be effectiveagainst E. coli in this study, but most gram-negative isolates arenonfermenters, a feature which imparts resistant to NF. NO, however, hasa broad range of antimicrobial, antifungal and antiviral activity.

Example 20 Nitric Oxide impregnation of Tracheal Tubes using ReducedPressure

A whole pack of individually-packaged Tracheal Tubes was placed in a NOchamber as described herein. The chamber was subjected to a reducedpressure (−10 psi) atmosphere for 5 minutes and was thereafter filled upwith 20,000 ppm NO during 16 hours, following general procedure IIdescribed hereinabove.

A NO-impregnated tube was thereafter cut into 1 cm sections which wereput into 2.5 cm of ddH₂O in screw-capped vials.

NO release from the NO-impregnated tube was assayed for nitrates andnitrites, as described hereinabove, using the Griess reagent, during 1week.

The following Tracheal tubes were tested: Mallinckrodt: Hi-Lo Trachealtube 6.5 mm ID ref No. 86110 (triangles) and Mallinckrodt: Hi-ContourTracheal tube 4.5 oral/nasal 6.2, 11 mm ID ref No. 107-45.

The data is presented in FIG. 16 and clearly show continuous release ofNO during the entire period.

Example 21 Nitric Oxide Impregnation of Wrapped Tampons

Four types of commercially available tampons were selected to determinetheir anti-infective efficacy after being charged with gaseous nitricoxide, according to embodiments of the present invention.

Table 19 presents the compositions of the tampons selected for testingin this example.

TABLE 19 A B C D Composition Rayon Rayon and/or 100% Rayon and/or cottonfiber elemental and/or cotton Cotton and/or chloride free Cotton fiberfiber polyester bleached Polysorbate thread rayon 20 Weight (grams) 1.53± 0.1 2.054 ± 0.1  1.54 ± 0.05 1.723 ± 0.1 Water 15 ± 1 29 ± 1 20 ± 1  32 ± 2 Absorption in 1 min (ml) Amount of 250 220 400 220 nitritesreached after 2 h (μmol)

Tampons were put in a petri dish in their individual packaging andimpregnated with nitric oxide using a 20,000 ppm cylinder, using generalprocedure I described hereinabove. Tampons were charged overnight (12-16hours) at a flow rate of 0.05 liter/minute. Control tampons were useddirectly out of their packaging.

Determination of Nitrites Released from Nitric Oxide ImpregnatedTampons:

Nitric oxide impregnated tampons were removed from their individualwrappers and cut in half into equally weighted pieces. Each piece wasplaced inside a separate 50 ml vial to which 25 ml of distilled water(dH₂O) was added. At time points of 10, 30, 60, and 120 minutes,nitrites were measured using Griess test, using absorbance at 543 nm,essentially as described hereinabove. The concentration of nitrites wasdetermined using a standard curve. The results are presented in Table 20below.

FIG. 17 presents a bar-graph showing the amount of nitric oxide releasedafter 30 minutes from four exemplary NO-impregnated tampons, as measuredby the respective total amount of nitrites released from the tamponsimpregnated with nitric oxide, wherein the letters under each bar denotethe tampon type presented in Table 19 hereinabove.

As can be seen in FIG. 17, all the tampon types sequestered nitric oxideas demonstrated by their ability to release nitric oxide once immersedin solution. During the two hours of immersion, nitric oxide wasreleased from the tampons at different rates. After 30 minutes ofimmersion in water, Tampon B released the highest amount of NO, at 100μmole nitrites per tampon, while tampon D released the lowest amount at14 μmole nitrites per tampon.

FIG. 18 presents comparative plots showing the accumulated of nitricoxide production during the first 5 hours for four types ofNO-impregnated tampons, as measured by the respective total accumulationof nitrites in water produced from tampons impregnated with nitricoxide, wherein nitrites released were calculated per 1 tampon.

As can be seen in FIG. 18, NO release from all tampons in water reacheda peak after 2 hours, with tampon B releasing NO at the highest rate,totaling about 196 μmole nitrite, as compared to the other tampons.Tampons C cumulatively released about 88 μmole nitrite each, tampon Areleased 55 μmole nitrite and tampon D released the lowest amount ofabout 32 μmole nitrite. All tampons released between 46-60% of theirnitric oxide respectively (measured as nitrites) within the first 30minutes, thus demonstrating a slower release rate.

Without being bound by any particular theory, the difference in releaserates is assumed to be a function of tampon composition, penetration ofNO through the packaging, absorption capacity, and other substancerelated factors. All four tampons tested reached a peak in theirrespective release profiles within the first three hours of immersion inwater. The total amount of nitric oxide released from a single tampon in25 ml water varied from 200 to 450 μmoles (reaching concentrations of 8to 18 mM). All four tampon brands tested released between 46-60% of thecharged NO within the first 30 minutes (out of 4 hours tested). Thus,some slow release was demonstrated, although it was more rapid than thatof other medical devices such as Foley catheters.

Anti-Infective Activity of Nitric Oxide Impregnated Tampons:

Yeast Preparation:

Candida albicans yeast culture was obtained from the American TypeCulture Collection (ATCC #14053). A starter was prepared by overnightgrowth of the colony taken from a plate of C. albicans in Difco™Sabouraud

Dextrose Broth (SAB) media at 30° C. Once turbid, the Optical Dispersionat 600 nm (OD₆₀₀) was measured and the culture was diluted with sterileSAB to give an OD₆₀₀ of 0.1, representing a culture containing 10⁶CFU/ml. From the culture of 10⁶ CFU/ml, two separate cultures containingyeast concentrations of 10¹ and 10² CFU/ml were prepared in sterile SABinside 1 L sterile containers.

Presence of C. albicans on the Surfaces of Charged Tampons:

In order to determine whether fungal growth could be prevented on thesurface of impregnated tampons, treated tampons were inoculated with C.albicans culture for 4 hours at 30° C. Growth was assessed by rollingthe treated tampons on a petri dish incubated overnight at 30° C. FIG.19 shows representative plates from each brand of tampon. No growth ofC. albicans in plates rolled with NO impregnated tampons was observed.In contrast, controls from all brands showed growth of C. albicans withtotal numbers of colonies on each plate being approximately 10 timesgreater for controls inoculated with 10⁵ CFU/ml than those inoculatedwith 10⁴ CFU/ml. Controls of all four brands of tampons showed similartotal numbers of colonies on corresponding plates.

Antimicrobial Activity of Impregnated Tampons against C. albicans and E.coli:

Tampons were aseptically removed from their individual wrappers andinserted into separate sterile 50 ml vials. The string of each tamponwas left hanging on the outside of each vial. Twenty-five (25) ml of C.albicans culture at either 10¹ or 10² CFU/ml was added to each vial andvortexed thoroughly. Vials were then incubated at 30° C. for 4 (10²CFU/ml) or 6 (10¹ CFU/ml) hours and thoroughly vortexed every 2 hours.Following incubation, aliquots (100 μl) were plated onto SAB agar petriplates and incubated overnight at 30° C. CFU were counted and calculatedto represent the CFU/ml.

FIG. 20 is a bar-graph showing the anti-infective activity ofNO-impregnated tampons, according to some embodiments of the presentinvention, comparing the growth of C. albicans in media after immersionof the tampons for 6 hours in suspension comprising 10¹ CFU/ml of C.albicans for NO-impregnated tampons and for untreated control tampons,wherein numbers represent viable counts of the triplicate CFUs, whitebars represent data from the control experiments and black barsrepresent data from the NO-impregnated tampons, with error barsrepresenting standard deviation.

As can be seen in FIG. 20, the effect of impregnated tampons on C.albicans growth was measured using various initial inoculums of yeast.Using an initial concentration of 250 CFU in total, tampon B has shownto eradicate all yeast, tampon C was shown to eradicate over 95% ofyeast compared to the control, tampon D was shown to have a 50%reduction over 4 hours, and tampon A did not have any effect compared tothe control tampons.

FIG. 21 is a bar-graph showing the anti-infective activity ofNO-impregnated tampons, according to some embodiments of the presentinvention, comparing the growth of C. albicans in media after immersionof the tampons for 4 hours in suspension comprising 10² CFU/ml of C.albicans for NO-impregnated tampons and for control untreated tampons,wherein numbers represent viable counts of the triplicate CFUs, whitebars represent data from the control experiments and black barsrepresent data from the NO-impregnated tampons, with error barsrepresenting standard deviation

As can be seen in FIG. 21, an effect similar to that described above hasbeen demonstrated when the media was inoculated with 2500 CFU in total.The only exception in results between the 10² and 10¹ inoculums was seenin the higher inoculi, tampon D did not appear to have any reductioncompared to the control, while it did have an anti-infective effectusing the lower inoculum of 10¹.

Table 20 summarizes the results of the abovementioned comparison of theobtained results.

TABLE 20 A B C D Amount of Second Highest Second Lowest nitrites lowest(196 highest (32 reached (55 μmoles) (88 μmoles) after 2 hours μmoles)μmoles) Slow release Yes (46% Yes (50% Yes (60% Yes (43% effect duringafter 30 after 30 after 30 after 30 5 hours minutes) minutes) minutes)minutes) Efficiency in No 100% 94% reduction 50% reduction controllingat 10¹ and at 10¹, none yeast infection 75% reduction at 10² at 10²

As can be seen in Table 20, the capacity of various tampons to absorbnitric oxide, according to a process embodiment of the presentinvention, and to release nitric oxide in an aqueous environment,according to other embodiments of the invention, has been demonstrated.Once immersed in an aqueous solution, tampons released nitric oxide,which was converted to its more stable metabolites, namely nitrites.Nitric oxide was released from the various brands of tampons atdifferent rates as seen in FIG. 18.

Further experiments were performed for determining the antibacterialeffect of NO-charged tampons, using 10³-10⁵ CFU/ml C. albicans and E.coli. The results are presented in Tables 21 and 22, respectively.

NO charged tampons were shown to eradicate 100% of C. albicans wheninoculated with 10³-10⁵ CFU/ml and incubated for 4 hours (Table 21). Incontrast, untreated tampons showed similar numbers of colonies,regardless of tampon type. Untreated tampons contained average numbersof colonies of 3×10³, 2×10⁴, and 2×10⁵ for staring concentrations of10³, 10⁴, and 10⁵, respectively.

NO charged tampons were shown to either reduce, or eradicate E. coliwhen inoculated with 10³-10⁵ CFU/ml after being incubated for 4 hours at37° C. (Table 22). Tampons B and D showed complete eradication of E.coli at concentrations 10³-10⁵ CFU/ml whereas tampon A eradicatedbacteria at concentrations of 10³ and 10⁴ CFU/ml, and showed asignificant reduction in colonies at 10⁵ CFU/ml. Untreated tamponscontained averages numbers of colonies of 3×10⁴, 4×10⁵, and 1×10⁶ forstaring concentrations of 10³, 10⁴ and 10⁵ CFU/ml, respectively.

TABLE 21 10⁵ 10³ 10⁴ Treat- Control * 10³ Treated Control * 10⁴ TreatedControl * 10⁵ ed A 1.3 ± 0.04 0 1.5 ± 0.09 0 1.0 ± 0.30 0 B 1.3 ± 0.20 01.5 ± 0.50 0 1.0 ± 0.20 0 C 1.7 ± 0.40 0 0.9 ± 0.30 0 1.1 ± 0.06 0 D 1.3± 0.01 0 0.6 ± 0.09 0 0.7 ± 0.20 0

TABLE 22 10³ 10⁴ 10⁵ Control * 10³ Treated * 10³ Control * 10⁴ Treated *10⁴ Control * 10⁵ Treated * 10⁵ A 31.0 ± 15 0 51 ± 18 0 8.5 ± 0.40 0.01± 0.01 B 20.0 ± 6.4 0 23 ± 10 0 8.5 ± 0.60 0 C 09.5 ± 3.0 0.06 ± 0.07 15± 13 0.1 ± 0.05 8.5 ± 0.04 0.30 ± 0.20 D 09.4 ± 1.8 0 43 ± 17 0 7.9 ±3.10 0

Inhibition of Bacterial Growth by Tampons Releasably Sequestering NO:

In order to check whether NO inhibits bacterial growth in the proximityof the devise, the clear zone surrounding the NO impregnated tampons wasmeasured. Charged tampons were cut lengthwise into two, then placed ontoan LB agar petri dish that had been inoculated with 200 μl of E. coli at10⁶ CFU/ml and incubated overnight at 37° C. Following incubation, theradius from the center of the tampon to the first viable colony wasmeasured.

Results, shown in FIG. 22 and Table 23, show that all charged tamponscreated a zone of bacterial growth inhibition with tampon B showing thehighest zone of inhibition with 4.2 cm to the first shown colony. In alluntreated tampons, no measureable zone of inhibition was observed.

TABLE 23 Zone of inhibition Control Treatment (cm) A 0 3.9 ± 0.4 B 0 4.2± 0.2 C 0 3.2 ± 0.7 D 0 3.8 ± 0.7

Inhibition of Bacterial Growth in a Vaginal Model:

NO-treated and control tampons were suspended inside sterile 250 mlErlenmeyer flasks containing LB, 2% (w/v) gelatin, and E. coli at 10⁶CFU/ml. Erlenmeyer flasks were incubated overnight at 37° C. The openingof an Erlenmeyer flask was utilized to model the shape of a vagina, asshown in the set-up illustration presented in FIG. 23.

FIG. 25 shows a picture comparing the effect of a NO-treated tampon Bagainst an untreated tampon when placed inside an Erlenmeyer flaskcontaining LB, 2% (w/v) gelatin, and E. coli at 10⁶ CFU/ml. It can bereadily seen that the NO treated tampon rendered the solid matrix insidethe flask much clearer than the untreated tampon, indicating either asignificant reduction or eradication of E. coli. In contrast, the matrixof the untreated tampon was evenly turbid throughout, indicatingconsiderable bacterial growth. Additionally, the clear matrix of theNO-treated tampon in FIG. 25 suggests that the NO released from thetampon (as NO₂ ⁻) diffused over a great enough distance to create alarge zone of inhibition in liquid.

The data presented herein corroborate the effectiveness of NO-treatedtampons as an antimicrobial delivery system inside the vagina, and henceas an effective treatment of, for example, vulvovaginal candidiasis(VVC), BC and/or RVVC.

Example 22 Nitric Oxide Impregnation of Unwrapped Tampons

Unwrapped tampons were treated with nitric oxide using general procedureI described hereinabove, except that the tampons were charged withnitric oxide outside their packaging material and additionalconcentrations of C. albicans were used during the experiment. Theresults are summarized in FIG. 25 and Tables 24 and 25.

FIG. 25 presents comparative plots of the total accumulation of nitritesproduced in water during 4 hours of immersion as a function of time, asmeasured by the Griess test from NO-treated and untreated tampons, alloutside their wrappers, wherein letters denote the type of tampon andthe nitrite levels are calculated per a single tampon.

Table 24 presents a comparative summary of tampons' ability to releaseNO and efficacy in controlling yeast growth. Table 25 presents acomparison of growth of C. albicans in media from tampons impregnatedwith nitric oxide without their wrappers versus media from controltampons, after immersion of the tampons for 4 hours in suspensioncomprising 10¹-10⁵ CFU/ml of C. albicans. The numbers represent theaverages and standard deviations of viable counts of the triplicateCFUs. “C” denotes negative control samples.

TABLE 24 A B C D Amount of nitrites reached 250 400 220 220 after 2hours μmoles μmoles μmoles μmoles Efficacy in controlling yeast 100%100% 100% 100% infection (10¹-10⁵ CFU/ml)

TABLE 25 Tampon Type A B C D Inocula C- C- C- C- (CFU/ml) C-avg stdevTest C-avg stdev Test C-avg stdev Test C-avg stdev Test 10¹ 30 0 0 35 70 20 14 0 35 7 0 10² 60 14 0 165 49 0 125 35 0 170 28 0 10³ 1780 212 01710 424 0 1345 7 0 2330 42 0 10⁴ 19050 4879 0 8600 3394 0 5750 919 015450 919 0 10⁵ 121000 21213 0 107500 6364 0 73500 23335 0 100000 296980

As can be seen in FIG. 25 and Tables 24 and 25, the charged tampons werevery effective in eradicating and/or controlling yeast growth. In fact,as demonstrated in Tables 24 and 25, all of the tampons charged withnitric oxide had zero yeast count after 4 hours of immersion in 10¹-10⁵CFU/ml yeast, indicating complete eradication of the yeast.

Example 23 Nitric Oxide Impregnation of Tampons

Wrapped and unwrapped tampons are treated with nitric oxide usinggeneral procedure II described hereinabove. As opposed to the prolongedtime (e.g., overnight) of exposure to nitric oxide required toimpregnate tampons with NO using general procedure I (see, Example 22hereinabove), only a few (e.g., 2-4) hours of exposure to nitric oxideare required using general procedure II.

Total accumulation of nitrites produced in water during 4 hours ofimmersion as a function of time is measured by the Griess test forNO-treated and untreated tampons. Nitrite levels of tampons impregnatedwith NO using general procedure II are higher than those impregnatedwith NO using general procedure I.

The tampons' efficacy in controlling yeast growth is determined bymeasuring growth of C. albicans in media from NO-treated and untreated,after immersion of the tampons for 4 hours in suspension comprising10¹-10⁵ CFU/ml of C. albicans.

Complete eradication of the yeast is observed in all NO-treated tampons.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

1.-117. (canceled)
 118. A process of preparing an article having gaseousnitric oxide sequestered therewithin, the process comprising: placing anarticle within a chamber; generating a reduced pressure in said chamber;and filling said chamber with a gaseous nitric oxide-containingenvironment, thereby preparing the article having gaseous nitric oxidesequestered therewithin.
 119. The process of claim 118, wherein saidgenerating said reduced pressure comprises reducing said pressure byfrom −1 psi to −50 psi.
 120. The process of claim 118, wherein at leasta portion of the article comprises a plurality of voids for sequesteringnitric oxide.
 121. The process of claim 118, wherein the article is amedical device.
 122. The process of claim 121, wherein said medicaldevice is an implantable device.
 123. The process of claim 121, whereinsaid medical device is a tampon.
 124. The process of claim 121, whereinsaid medical device comprises a polymeric material.
 125. The process ofclaim 118, wherein said article is selected from the group consisting ofa packaged article and a bare (unpackaged) article.
 126. The process ofclaim 125, wherein said packaged article comprises a gas-permeablepackage.
 127. An article having a gaseous nitric oxide sequesteredtherewithin, prepared by the process of claim
 118. 128. The article ofclaim 127, further comprising an enclosure.
 129. The article of claim127, comprising at least 1 ppm per cm³ nitric oxide sequestered therewithin.
 130. The article of claim 127, wherein said sequestered nitricoxide is releasable is an aqueous solution during a time period thatranges from 1 hour to 1 month.
 131. An article having sequesteredtherewithin at least 1 ppm nitric oxide per cm³ and comprising less than1 ppm per cm³ nitrogen-containing and/or oxygen containing reactivespecies.
 132. The article of claim 131, having sequestered therein from1 ppm to 200 ppm per cm³ nitric oxide.
 133. The article of claim 131,wherein said sequestered nitric oxide is releasable in an aqueoussolution during a time period that ranges from 1 hour to 1 month. 134.The article of claim 131, further comprising an enclosure.
 135. Thearticle of claim 127, being a medical device.
 136. The article of claim135, wherein said medical device is selected from the group consistingof an indwelling catheter and a tracheal tube.
 137. The article of claim135, wherein said medical device is a tampon.
 138. A tampon havingsequestered therein gaseous nitric oxide.
 139. A process of preparing atampon having sequestered therein nitric oxide, the process comprising:exposing a tampon to gaseous nitric oxide-containing environment,thereby preparing the tampon having sequestered therein nitric oxide.140. The process of claim 139, wherein said exposing comprises: placingthe tampon is a chamber; and filling the chamber with said nitricoxide-containing environment.
 141. The process of claim 140, furthercomprising, prior to said filling, generating a reduced pressure in saidchamber.
 142. A method of treating a vaginal medical condition in asubject in need thereof, the method comprising placing a tampon havinggaseous nitric oxide sequestered therein in a vagina of the subject.143. A method of treating a vaginal medical condition, the methodcomprising intravaginally administering to a subject in need thereofgaseous nitric oxide.
 144. The method of claim 143, wherein saidintravaginally administering gaseous nitric oxide comprises placing atampon having gaseous nitric oxide sequestered therein.
 145. A processof preparing a packaged article, wherein the packaged article comprisesa gas-permeable package, the process comprising: exposing a packagedarticle to a gaseous nitric oxide-containing environment, therebypreparing the packaged article.
 146. The process of claim 145, whereinsaid exposing comprises: placing said packaged article in a chamber; andfilling the chamber with said nitric oxide-containing environment. 147.The process of claim 146, further comprising, prior to said filling,sealing said chamber.
 148. The process of claim 146, further comprising,prior to said filling, generating a reduced pressure in said chamber.149. The process of claim 145, wherein the packaged article has gaseousnitric oxide sequestered within the article.
 150. A process of preparinga packaged article, wherein the packaged article comprises a non-gaspermeable enclosure, the process comprising: positioning an intactarticle within said non-gas permeable enclosure, to thereby obtain a nongas-permeable enclosure having said article disposed therewithin;exposing said enclosure with a gaseous nitric oxide-containingenvironment, so as to introduce into said enclosure said nitricoxide-containing environment; and sealing said enclosure, therebypreparing the packaged article.
 151. The process of claim 150, whereinsaid exposing comprises: placing said enclosure in a chamber; andfilling said chamber with said gaseous nitric oxide-containingenvironment.
 152. The process of claim 151, further comprising, prior tosaid filling, sealing said chamber.
 153. The process of claim 152,further comprising, prior to said filling, reducing a pressure in saidchamber.
 154. The process of claim 150, wherein the packaged article hasgaseous nitric oxide sequestered within the article.
 155. A packagedarticle prepared by the process of claim
 145. 156. A packaged articleprepared by the process of claim
 150. 157. A package comprising: amaterial configured to form an enclosure; a article disposed within theenclosure; and a gaseous nitric oxide-containing environment within saidenclosure.
 158. The package of claim 157, wherein said package is anon-gas permeable package.
 159. The package of claim 157, wherein saidenclosure is a sealed enclosure.
 160. The package of claim 157, whereinsaid environment is an ambient environment.
 161. The package of claim157, wherein said environment comprises gaseous nitric oxide in anamount sufficient to sterilize said article and an interior of saidenclosure.
 162. The package of claim 157, wherein said article hasgaseous nitric oxide sequestered therewithin.
 163. The package of claim162, wherein said gaseous nitric oxide sequestered in said article isreleasable in an aqueous solution during at least 1 minute.
 164. Acharging device comprising: a chamber comprising an inlet for receivinga gaseous nitric-oxide containing environment and an outlet forreleasing said gaseous nitric-oxide containing environment; and anarticle disposed within the chamber.
 165. The device of claim 164,further comprising an outlet for generating a reduced pressure in saidchamber.
 166. A charging device comprising: a sealed chamber having areduced pressure therewithin; and an article disposed within thechamber.
 167. The device of claim 166, wherein the chamber furthercomprises an outlet for generating said negative pressure within saidchamber.
 168. The device of claim 167, further comprising an inletconfigured for receiving a gaseous nitric oxide-containing environmentand an outlet for releasing said gaseous nitric oxide-containingenvironment.